EP4203999A1 - Acides nucléiques codant pour un polypeptide comprenant une région fc modifiée d'une igg1 humaine et au moins un antigène hétérologue - Google Patents

Acides nucléiques codant pour un polypeptide comprenant une région fc modifiée d'une igg1 humaine et au moins un antigène hétérologue

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Publication number
EP4203999A1
EP4203999A1 EP21769694.7A EP21769694A EP4203999A1 EP 4203999 A1 EP4203999 A1 EP 4203999A1 EP 21769694 A EP21769694 A EP 21769694A EP 4203999 A1 EP4203999 A1 EP 4203999A1
Authority
EP
European Patent Office
Prior art keywords
seq
nucleic acid
region
modified
rbd
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21769694.7A
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German (de)
English (en)
Inventor
Linda Gillian Durrant
Mireille VANKEMMELBEKE
Victoria BRENTVILLE
Rachael METHERINGHAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Scancell Ltd
Original Assignee
Scancell Ltd
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Publication date
Priority claimed from GBGB2013385.6A external-priority patent/GB202013385D0/en
Priority claimed from GBGB2101435.2A external-priority patent/GB202101435D0/en
Application filed by Scancell Ltd filed Critical Scancell Ltd
Publication of EP4203999A1 publication Critical patent/EP4203999A1/fr
Pending legal-status Critical Current

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001156—Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]
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    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001188—NY-ESO
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    • A61K39/001192—Glycoprotein 100 [Gp100]
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    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
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    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to nucleic acids and peptides encoded by those nucleic acids.
  • the peptides comprise a modified IgGl Fc region and one or more heterologous epitopes, which may be B- or T-cell epitopes.
  • the nucleic acids and peptides of the invention can be used as a vaccine that stimulates high avidity CD8 T cells, Thl CD4 T-cells and strong antibody responses.
  • stimulation of potent cellular immunity including both CD4 and CD8 T-cell responses is vital.
  • stimulation of both cellular immunity and neutralising antibodies are important.
  • the immune system is a powerful defence mechanism that can be employed to combat harmful pathogens and malignancies.
  • the adaptive immune system in particular can be educated through vaccination to target altered, mutated or over-expressed self-antigens that are characteristic of malignant disease and invading foreign pathogens such as viruses.
  • immune surveillance The concept that the immune system can recognise and eliminate cancerous cells is referred to as immune surveillance [1, 2], Immunological differences between cancer and self can be detected naturally and lead to elimination of cancerous cells in many individuals. This theory suggests that the immune system is naturally capable of detecting and eliminating cancer cells.
  • mice Studies in immunocompetent mice show rejection of tumours and the protection gained can be adoptively transferred to mice via transfer of the T cells [3].
  • knockout models showed that interferon gamma ( I FNy) and lymphocytes are important in reducing the incidence of carcinogen-induced sarcoma and spontaneous epithelial carcinomas [4]
  • Patients with spontaneously regressing melanoma showed signs of tumour-specific clonal T cell expansion providing evidence of immune surveillance [5]
  • Recent work has demonstrated that the mutations accrued by tumours during the transformation process can generate neo-antigens that can be efficiently targeted by T cells [6]
  • This work all highlights that the fact that the immune system is capable of distinguishing between tumour and self but cancer still develops in many immunologically healthy individuals.
  • tumours do not present the correct environmental conditions to prime an effective immune response.
  • TLRs Toll-like receptors
  • DAMPS damage-associated molecular pattern molecules
  • Viruses can be broadly classified as being either 'non-enveloped' or 'enveloped'. Functionally, the viral envelope enables entry of a virus into its host. Viral glycoproteins on the envelope surface recognise and bind to receptor sites on the host's cell membrane. This leads to fusion of the viral envelope with the host's membrane, entry and release of the viral genome and infection of the host. Early in a viral infection both CD4 and CD8 T-cell responses are generated to locate and kill virally infected cells and prevent further replication of the virus. Later in the infection virus neutralising antibodies (VNabs) are produced to help prevent reinfection. The combination of memory T-cell responses and VNabs are both important in preventing new infections to the same or related viruses.
  • VNabs virus neutralising antibodies
  • Vaccines need to stimulate an immune response prior to exposure to the virus/pathogen and are designed to enable the host to respond quickly and efficiently to remove low viral load and to prevent any morbidity associated with the virus.
  • a vaccine needs to stimulate high avidity T-cells that recognise low antigen load and neutralising antibodies which prevent viral entry into a cell.
  • T-cells can recognise viral proteins as they will all be presented in the context MHC on the cell surface of infected cells.
  • the neutralising antibodies need to bind to the viral proteins which contact receptors on host cells and allow their entry into the cell.
  • the most efficient viral vaccines are attenuated viruses which stimulate potent T cells and antibody responses but are associated with a low morbidity.
  • Attenuated virus vaccines There are already several licensed attenuated virus vaccines including smallpox, measles and polio. However, many viruses have evolved to evade immune recognition and are therefore not suitable as attenuated viral vaccines. This may be overcome by using an inactivated virus which is achieved using chemicals such as formaldehyde or heat inactivation. Such vaccines can stimulate antibody responses but require large quantities of the virus/pathogen and are very poor at stimulating high avidity T-cells response. A similar approach is to use virus like particles which assemble and look like viruses but cannot replicate. Again, these vaccines induce strong antibody responses and have been licensed to prevent HPV infection. An alternative to an attenuated virus is to use a hybrid virus.
  • Viruses such as measles or adenovirus are genetically modified to produce proteins from a heterologous virus. These viruses are weakened or disabled so that they cannot cause disease; they can either still replicate within cells or they have had genes deleted that render them incapable of replicating.
  • the viral vectors tend to have a good safety profile and an Ebola vaccine has recently been approved.
  • the existence of any pre-existing immunity to the viral vector may impact their effectiveness and limits the ability to boost waning immune responses. They can also be only used for a single virus as once an immune response to the carrier virus has been established they cannot be used as a carrier for a new virus.
  • viral vaccines can generate a high level of protein expression, inducing a strong antibody response. This can also be achieved using protein vaccines.
  • protein vaccines also require adjuvants to simulate a potent immune response; in addition multiple doses are often required. Both protein and heterologous viral vaccines produce high levels of antigen but this stimulates low avidity T cell responses that will only kill cells with a high viral load which is dangerous as these cells may be lysed by the virus and spill large quantities of virus into the host before the low avidity T-cell has had time to react.
  • US7067110B1 discloses the use of an Fc-antigen fusion protein whereby whole antigen or antigen domains are fused to the hinge-CH2-CH3 domains of an antibody can enhance both antibody and cellular immunity.
  • WO 2002/058728 discloses that targeting FcyRI with a polypeptide human IgGl Fc fused to an antigen can stimulate high avidity T-cell responses.
  • WO2008/116937 discloses a nucleic acid which comprises a non-specific promoter and at least one sequence that encodes a recombinant heavy chain of an immunoglobulin molecule, wherein the heavy chain has at least one heterologous T cell epitope therein such that the heavy chain cannot take its native conformation when the nucleic acid is expressed.
  • the present invention provides a nucleic acid which encodes a polypeptide comprising:
  • the modified Fc region comprises at least the part of Fc that is capable of binding to CD64, (b) at least one residue of the modified Fc region is modified to the corresponding residue from a mouse lgG3 antibody and (c) the modified Fc region has enhanced avidity for Fc-gamma receptor (FcyR) when compared to the corresponding wildtype Fc region.
  • FcyR Fc-gamma receptor
  • the present invention provides a nucleic acid which encodes a polypeptide comprising:
  • the modified Fc region comprises at least the part of Fc that is capable of binding to TRIM21, (b) at least one residue of the Fc region is modified to the corresponding residue from a mouse lgG3 antibody and (c) the modified Fc region has enhanced avidity for Fc-gamma receptor (FcyR) when compared to the corresponding wildtype Fc region.
  • FcyR Fc-gamma receptor
  • the present invention provides a nucleic acid which encodes a polypeptide comprising:
  • the modified Fc region comprises at least the part of Fc that is capable of binding to CD64 and/or TRIM21, (b) at least one residue of the Fc region is modified to the corresponding residue from a mouse lgG3 antibody and (c) the modified Fc region has enhanced avidity for Fc- gamma receptor (FcyR) when compared to the corresponding wildtype Fc region.
  • FcyR Fc- gamma receptor
  • the present invention provides a vector comprising the nucleic acid of the first aspect.
  • the present invention provides a polypeptide encoded by the nucleic acid of the first aspect or a vector of the second aspect.
  • the inventors have unexpectedly found that transferring certain mouse lgG3 (mlgG3) Fc residues into the hlgGl Fc region of an antigen-Fc fusion protein improves the immunogenicity of the antigen.
  • MlgG3 is the only isotype among the mlgGs that forms non-covalent oligomers, strongly influencing their biological activity [10], and increasing functional affinity to polyvalent antigens.
  • Fc modified polypeptide of the invention binds initially in a monomeric form, it cannot bind to the low affinity FcRIlb and FcRIIIb inhibitory receptors which would result in inhibition of immune responses.
  • the creation of an improved vaccine, with enhanced immunogenicity, through establishing intermolecular cooperativity binding, may lead to superior clinical utility.
  • the modified Fc region may have avidity for Fc-gamma receptor (FcyR), preferably FcyRI, that is enhanced by at least about 10% when compared to a corresponding wildtype human IgGl Fc region.
  • the modified Fc region may have avidity for Fc-gamma receptor (FcyR), preferably FcyRI, that is enhanced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% when compared to a corresponding wildtype human IgGl Fc region.
  • the polypeptide of the present invention preferably has enhanced immunogenicity and/or non- covalent oligomerisation when compared to a corresponding peptide comprising an unmodified wildtype human IgGl region and at least one heterologous antigen.
  • the immunogenicity and/or non-covalent oligomerisation of the polypeptide of the present invention may be enhanced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% when compared to a corresponding polypeptide comprising an unmodified wildtype human IgGl region and at least one heterologous antigen.
  • the modified Fc region comprises the part of Fc that binds to CD64 and/or TRIM21. It may comprise CH2 and CH3, and optionally may further comprise the hinge region.
  • One or more residues of the human IgGl CH2 and/or the CH3 domain may be replaced with the corresponding residues from the mouse lgG3 CH2 and/or CH3 domain.
  • the at least one residue of the Fc region is selected from the CH2 and/or the CH3 domains. In some aspects, the at least one residue is selected from the CH2 domain. In some aspects, the at least one residue is selected from the CH3 domain.
  • the 23 residues are: N286T, K288W, K290Q, A339P, Q.342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q.362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.
  • These residues are required for increased non-covalent oligomerisation through intermolecular cooperativity due to the combined effect of directly interacting as well as conformational residues, the latter potentially creating a permissive framework. Further details of the modification to the Fc region are set out in PCT/EP2020/071724, the contents of which are fully incorporated by reference.
  • the Fc region of a human IgGl may have modifications to one or more of the following residues of the Fc region: N286, K288, K290, A339, Q.342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q.362, K370, G371, Y373, P374, S375, D376, A378.
  • the modifications may be one or more of N286T, K288W, K290Q, A339P, Q.342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q.362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.
  • the modified Fc region of a human IgGl may comprise modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 residues selected from positions N286, K288, K290, A339, Q.342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378.
  • the modified Fc region of a IgGl antibody comprises modifications at all 23 residues.
  • the modified Fc region of a human IgGl may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 modifications selected from N286T, K288W, K290Q, A339P, Q.342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q.362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.
  • the modified Fc region of a IgGl antibody comprises all 23 modifications.
  • the modified Fc region may comprise modifications to one or more of the following residues of the Fc region: N286, K288, K290, Q.342, P343, E345, L351, T359, N361, Q.362, G371, P374, S375, D376, A378.
  • the modifications may be one or more of N286T, K288W, K290Q, Q.342R, P343A, E345T, L351I, T359S, N361K, Q.362K, G371N, P374S, S375E, D376A, A378S.
  • the modified Fc region may comprise modifications at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 residues selected from positions N286, K288, K290, Q.342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378.
  • the modified Fc region of a human IgGl antibody comprises modifications at all 15 residues.
  • the modified Fc region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 modifications selected from N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.
  • the modified Fc region comprises all 15 modifications.
  • the modified Fc region may comprise modifications to one or more of the following residues of the Fc region: Q342, P343, E345, N361, Q362, P374, D376.
  • the modifications may be one or more of Q342R, P343A, E345T, N361K, Q362K, P374S, D376A.
  • the modified Fc region may comprise modifications at 1, 2, 3, 4, 5, 6 or 7 residues selected from positions Q342, P343, E345, N361, Q362, P374, D376.
  • the modified Fc region comprises modifications at all 7 residues.
  • the modified Fc region may comprise 1, 2, 3, 4, 5, 6 or 7 modifications selected from Q342R, P343A, E345T, N361K, Q362K, P374S, D376A. Preferably, the modified Fc region comprises all 7 modifications.
  • the modified Fc region may comprise the amino acid sequence provided in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity to SEQ. ID NO: 1.
  • SEQ ID NO: 1 is the amino acid sequence of an exemplary modified Fc region, "ivl" (see Table 4).
  • the structure of the polypeptide of the present invention may be of an antibody heavy chain sequence or substantial portion thereof.
  • the structures and locations of immunoglobulin domains may be determined by reference to http://www.imgt.org/.
  • residue numbering refers to the standardised IMGT system for the numbering of antibody sequences, as disclosed in Lefranc et al., 2009 [17], Other suitable numbering systems are known to the skilled person. Other suitable numbering systems may be used to identify corresponding residues between the modified human IgGl antibody - antigen fusion protein thereof. Any numbering system which allows identification of corresponding residues is suitable for use with the present invention.
  • the numbering system used herein is not limiting on the scope of the invention, but is used simply to identify the relevant residues which may be modified.
  • the term "corresponding residue” is intended to mean the residue in the equivalent position, structurally or functionally, in the two or more antibodies or antigen-binding fragments thereof that are being compared. In some cases, corresponding residues may be identified by sequence alignment. In some cases, corresponding residues may be identified by structural comparison.
  • the polypeptide may comprise at least 10 amino acid residues of an Fc-region, at least 20 amino acid residues of an Fc-region, at least 30 amino acid residues of an Fc-region, at least 40 amino acid residues of an Fc-region, at least 50 amino acid residues of an Fc-region, at least 75 amino acid residues of an Fc-region, at least 100 amino acid residues of an Fc-region, at least 200 amino acid residues of an Fc-region, at least 300 amino acid residues of an Fc-region, at least 400 amino acid residues of an Fc-region or at least 500 amino acid residues of an Fc-region.
  • the polypeptide comprises the entire Fc-region of human IgGl.
  • the at least one heterologous antigen may be linked (directly or via a linker) to the N-terminus of the modified human IgGl Fc region. It is less preferred if the at least one heterologous antigen is linked (directly or via a linker) to the C-terminus of the modified human IgGl Fc region.
  • the heterologous antigen is a polypeptide, it may be linked to the modified Fc region at the N- or, more preferably, the C-terminus of the heterologous antigen.
  • This format of polypeptide of the invention may be used where the heterologous antigen is a relatively large molecule, such as a viral or bacterial protein or immunogenic fragment thereof.
  • the C-terminus of the heterologous antigen is linked, optionally via a linked, to the N terminus of the modified Fc region.
  • the at least one heterologous antigen comprises one or more epitopes selected from the epitopes set out in any one of Figures 28-33.
  • the at least one heterologous antigen comprises one or more epitopes selected from the epitopes set out in Table 2 or Table 3.
  • the at least one heterologous antigen comprises one or more epitopes selected from:
  • the at least one heterologous antigen comprises one or more epitopes selected from:
  • the at least one heterologous antigen comprises one or more epitopes selected from:
  • the at least one heterologous antigen comprises one or more epitopes selected from: (a) LLMWITQCF (SEQ ID NO: 35);
  • the at least one heterologous antigen comprises one or more epitopes selected from:
  • the at least one heterologous antigen comprises one or more epitopes selected from:
  • immunogenic fragment is a portion of an antigen or protein that is smaller than an entire antigen or protein and is capable of eliciting a humoral and/or cellular immune response specific to that fragment in a host animal (e.g., a human). Fragments of the protein can be produced using techniques known in the art, such as recombination, by proteolytic digestion, or by chemical synthesis. An internal or terminal fragment of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid encoding a polypeptide.
  • Linker sequences are usually flexible, in that they are made up primarily of amino acids such as glycine, alanine and serine, which do not have bulky side chains likely to restrict flexibility.
  • linkers with greater rigidity may be desirable. Usable or optimum lengths of linker sequences may be easily determined. Often the linker sequence will be less than about 12, such as less than 10, or from 2-10 amino acids in length, The linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length.
  • linkers examples include, but are not limited to: GGGGS (SEQ ID NO: 106), GGGSG (SEQ ID NO: 107), GGSGG (SEQ ID NO: 108), GSGGG (SEQ ID NO: 109), GSGGGP (SEQ ID NO: 110), GGEPS (SEQ ID NO: 111), GGEGGGP (SEQ ID NO: 112), GGEGGGSEGGGS (SEQ ID NO: 113) and GGGSGGGG (SEQ ID NO: 114).
  • GGGGS SEQ ID NO: 106
  • GGGSG SEQ ID NO: 107
  • GGSGG SEQ ID NO: 108
  • GSGGG SEQ ID NO: 109
  • GSGGGP SEQ ID NO: 110
  • GGEPS SEQ ID NO: 111
  • GGEGGGP SEQ ID NO: 112
  • GGEGGGSEGGGS SEQ ID NO: 113
  • GGGSGGGGGG SEQ ID NO: 114
  • Additional linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 115), TVLRT (SEQ ID NO: 116), TVSSAS (SEQ ID NO: 117) and TVLSSAS (SEQ ID NO: 118).
  • a preferred linker used in the present invention is the Ig hinge.
  • the polypeptide of the present invention may comprise an antibody variable region into which the or each heterologous antigen is inserted or substituted.
  • the polypeptide of the present invention may comprise a human IgGl heavy chain comprising the modifications in the Fc region thereof. It is preferred if the or each heterologous antigen is substituted into one or more of the CDRs of the variable region. Although all CDRs can be used for substitution for a heterologous antigen, a preferred CDR is CDR3. This format of polypeptide of the invention may be used where the heterologous antigen is, for example, a cancer antigen.
  • the antibody variable may be a heavy chain variable region comprising the following heterologous antigens substituted into the CDR1, CDR2 and CDR3 respectively:
  • GTGRAMLGTHTMEVTVYH (SEQ ID NO: 29), SVYDFFVWL (SEQ ID NO: 30) and VPLDCVLYRYGSFSVTLDIVQG (SEQ ID NO: 32); or
  • LLMWITQCF (SEQ ID NO: 35), SLLMWITQC (SEQ ID NO: 36) and PESRLLEFYLAMPFATPMEAELARRSLAQ (SEQ ID NO: 37).
  • polypeptide encoded by the nucleic acid of the invention may comprise the amino acid sequence provided in SEQ ID NO: 2 or in SEQ ID NO: 3.
  • SEQ ID NO: 2 and SEQ ID NO: 3 are the amino sequences of the whole antibody heavy chain encoded by the iSCI Blplus (see Figure 31) and iSCI B2 (see Figure 33) vectors respectively.
  • the heterologous antigen may be the N protein of a coronavirus or an immunogenic fragment thereof.
  • the N protein is from SARS-CoV-2.
  • the N protein may be from lineage A Wuhan strain SARS-CoV-2, B.1.351 variant SARS-CoV-2 or B.1.617.2 variant SARS-CoV-2.
  • the N protein may comprise the amino acid sequence provided in SEQ ID NO:4 (Wuhan strain).
  • the N protein may comprise the amino acid sequence provided in SEQ ID NO:5 (B.1.351 variant).
  • the N protein may comprise the amino acid sequence provided in SEQ ID NO:26 (B.l.1.7 variant).
  • the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided in SEQ ID NO: 6.
  • SEQ ID NO: 6 is the amino acid sequence of the N protein (Wuhan strain) fused to the ivl modified Fc region encoded by the "SN15" vector (see Figure 27).
  • the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided in SEQ ID NO: 7.
  • SEQ ID NO: 7 is the amino acid sequence of the N protein (B.1.351 variant) fused to the ivl modified Fc region encoded by the "SN17" vector (see Figure 48).
  • the polypeptide encoded by the nucleic acid of the invention may comprise the amino acid sequence provided in SEQ ID NO: 27.
  • SEQ ID NO: 27 is the amino acid sequence of the N protein (B.l.1.7 variant) fused to the ivl modified Fc region encoded by the "SN16" vector (see Figure 47).
  • the nucleic acid of the invention may be provided in combination (separately or linked) with a second nucleic acid encoding a second polypeptide comprising at least one heterologous antigen.
  • the second polypeptide may be an antibody light chain.
  • the light chain may have one or more heterologous antigens inserted or substituted therein.
  • the or each heterologous antigen may be substituted into one or more of the CDRs of the antibody light chain.
  • a preferred CDR is CDR3.
  • the antibody light chain encoded by the second nucleic acid may comprise the following heterologous antigens substituted into the CDR1, CDR2 and CDR3 respectively: WNRQLYPEWTEAQRLD (SEQ ID NO: 31), ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 33) and QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 34).
  • the antibody light chain encoded by the second nucleic acid may comprise the sequence PGVLLKEFTVSGNILTIRLTAADHR (SEQ ID NO: 38) substituted into the CDR2.
  • the antibody light chain encoded by the second nucleic acid may comprise the amino acid sequence provided in SEQ ID NO: 10 or SEQ ID NO: 11.
  • SEQ ID NOs: 10 and 11 are the amino acid sequences of the antibody light chains encoded in the iSCI Blplus ( Figure 31) and iSCIB2 ( Figure 33) vectors respectively.
  • the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided in SEQ ID NO: 2 and the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 10.
  • the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 3 and the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 11.
  • the second nucleic acid encodes the receptor binding domain of SARS-Cov-2.
  • the receptor binding domain may comprise the amino acid sequence provided in SEQ ID NO: 8 (Wuhan strain RBD).
  • the receptor binding domain may comprise the amino acid sequence provided in SEQ ID NO: 9 (B.1.351 variant RBD).
  • the receptor binding domain may comprise the amino acid sequence provided in SEQ ID NO: 28 (B.l.1.7 variant RBD).
  • the polypeptide encoded by the nucleic acid of the invention comprises the amino acid sequence provided in SEQ ID NO: 6 and the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided in SEQ. ID NO: 8.
  • the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 7 and the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided in SEQ ID NO: 9.
  • the polypeptide encoded by the nucleic acid may comprise the amino acid sequence provided in SEQ ID NO: 27, and the second nucleic acid encodes a receptor binding domain comprising the amino acid sequence provided in SEQ ID NO: 28.
  • the polypeptide encoded by the nucleic acid sequence of the invention may comprise an amino acid sequence that is at least 90% identical to any one of the above recited sequences.
  • the polypeptide encoded by the nucleic acid sequence may comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the above recited sequences.
  • the present invention provides one or both of the amino acid sequences disclosed in Figures 27, 28, 29, 30, 31, 32, 33, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 47, 48, 54, 55, 56, 57, 58 or 59, respectively.
  • Preferred polypeptides in accordance with the invention are set out in Figure 12b (optionally in combination with Figure 12a), Figure 13b (optionally in combination with Figure 13a), Figure 14b (optionally in combination with Figure 14a), Figure 15b (optionally in combination with Figure 15a), Figure 27b (optionally in combination with Figure 27a), Figure 29a (optionally in combination with Figure 29b), Figure 31a (optionally in combination with Figure 31b) and Figure 33b (optionally in combination with Figure 33a).
  • Particularly preferred sequences are disclosed in Figures 27, 31, 33, 48 and 54 to 59.
  • the present inventors have shown that immunisation with SN15, encoding lineage A Wuhan strain N protein and RBD, gives strong VNAbs against this strain but also cross reacts with B.1.351 and B.1.617.2 variant RBDs.
  • the inventors have shown that immunisation with SN17, encoding B.1.351 variant N protein and RBD, gives strong VNAbs against this strain but also cross reacts with lineage A Wuhan strain and B.1.617.2 variant RBDs.
  • the avidity of a polypeptide of the invention comprising a modified Fc region and the corresponding polypeptide comprising a wildtype Fc region may be determined by Surface Plasmon Resonance (e.g.
  • Biacore 3000/ T200, GE Healthcare for example by injecting increasing concentrations (0.3nmol/L-200nmol/L) of a polypeptide of the invention across a CM5 chip comprising an appropriate ligand (such as FcyRI) and fitting the data to an appropriate binding model using appropriate software (e.g. BIAevaluation 4.1).
  • a polypeptide of the invention for example by injecting increasing concentrations (0.3nmol/L-200nmol/L) of a polypeptide of the invention across a CM5 chip comprising an appropriate ligand (such as FcyRI) and fitting the data to an appropriate binding model using appropriate software (e.g. BIAevaluation 4.1).
  • BIAevaluation 4.1 e.g. BIAevaluation 4.1
  • the polypeptide of the invention comprising a modified Fc region shows greater functional affinity to ligand than the corresponding polypeptide having the wildtype Fc when the Surface Plasmon Resonance data indicates that the polypeptide of the invention binds more tightly to the ligand-coated CM5 chip.
  • the ligands may comprise Fc receptor, particularly Fey receptor. All of the Fey receptors (FcyR) belong to the immunoglobulin superfamily and are the most important Fc receptors for inducing phagocytosis of opsonized (marked) microbes.
  • FcyRI binds to IgG more strongly than FcyRII or FcyRIII does.
  • FcyRI also has an extracellular portion composed of three immunoglobulin (Ig)-like domains, one more domain than FcyRII or FcyRIII has. This property allows FcyRI to bind a sole IgG molecule (or monomer), but all Fey receptors must bind multiple IgG molecules within an immune complex to be activated.
  • a preferred receptor is FcyRI (CD64).
  • a preferred receptor is TRIM21.
  • the polypeptides of the invention may be capable of binding to CD64 and/or TRIM21.
  • TRIM21 is the cytosolic antibody receptor and E3 ubiquitin ligase. It detects antibody inside cells and mediates their rapid proteosomal degradation. If antibodies are modified to express T cell epitopes within their variable regions or heterologous antigens are linked to Fc and are administered via a DNA plasmid which can directly transduce antigen presenting cells, the protein would be translated within the cells and be targeted by TRIM21.
  • the Biacore CM5 chip coated with an anti-his antibody, comprises carboxymethylated dextran covalently attached to a gold surface.
  • Molecules are covalently coupled to the sensor surface via amine, thiol, aldehyde or carboxyl groups. Interactions involving small organic molecules, such as drug candidates, through to large molecular assemblies or whole viruses can be studied.
  • a high binding capacity gives a high response, advantageous for capture assays and for interactions involving small molecules.
  • High surface stability provides accuracy and precision and allows repeated analysis on the same surface.
  • Other suitable chips are known to the skilled person and the Surface Plasmon Resonance protocols can be adapted by standard techniques known in the art.
  • the immunogenicity of a polypeptide in accordance with the invention may be determined.
  • the improved properties of the polypeptide may be measured relative to the corresponding properties of a corresponding polypeptide that does not comprise the modified residues in the Fc-region. Since the improved functional properties are a relative measure, the precise method used to determine the immunogenicity, or any other functional property of the polypeptide of the invention, does not affect the relative change in that functional property.
  • polypeptide of the invention Whilst not wishing to be bound by theory, the ability of the polypeptide of the invention to provide enhanced immunogenicity may be a direct consequence of the modified human IgGl Fc binding to cell surface receptor.
  • the polypeptide and/or nucleic acid of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the nucleic acid/polypeptide.
  • Polypeptides of the invention comprise at least one heterologous antigen.
  • heterologous antigen is intended to mean an antigen which is heterologous to the modified Fc region.
  • the antigen may be a T cell antigen or a B cell antigen.
  • Some polypeptides in accordance with the invention comprise both T and B cell antigens.
  • the antigen may be comprised in a relatively large molecule, such as a viral or bacterial protein or immunogenic fragment thereof. Alternatively, it may be an amino acid sequence making up the antigen or the epitope within the antigen.
  • the antigen may be from a cancer or may be from an infectious disease. In some aspects the antigen is from a cancer. In some aspects the antigen is from an infectious disease.
  • the antigen may stimulate high avidity CD8 T cells, Thl CD4 T-cells and/or strong antibody responses. Where the invention is to be used as a cancer vaccine, stimulation of potent cellular immunity including both CD4 and CD8 T-cell responses is vital. Where the invention is to be used as a vaccine for infectious disease, stimulation of both cellular immunity and neutralising antibodies is important.
  • coronaviruses are members of the subfamily Coronavirinae (family Coronaviridae; order Nidovirales), which are classified into four genera, Alphacoronavirus (alpha-CoV), Betacoronavirus (beta-CoV), Gammacoronavirus (gamma-CoV), and Deltacoronavirus (delta-CoV) [18, 19], Gamma-CoV and delta-CoV generally infect birds, although some can cause infection in mammals.
  • the alpha-CoV and beta-CoV viruses are known to infect and cause disease in both humans and animals.
  • the SARS-CoV (beta-CoV), 229E (alpha-CoV), HKU1 (beta-CoV), NL63 (alpha-CoV) and OC43 (beta-CoV) viruses can all cause infections in humans [18], typically causing upper respiratory infections and some relatively minor symptoms [20].
  • the beta-CoV are the most pathogenic viruses in humans, this group also includes SARS-CoV-2, MERS-CoV, and SARS-CoV [18, 21, 22]; all have caused outbreaks in the 21 st century.
  • SARS-CoV-2 shows the greatest homology with SARS-CoV, demonstrating 79% genetic similarity [23], SARS-CoV-2 is most similar to the bat coronavirus RaTG13, with 98% similarity [24],
  • the genome of CoVs is a single-stranded positive-sense RNA (+ssRNA) with 5'-cap structure and 3'- poly-A tail.
  • the genomes of RNA viruses are typically less than 10 kb in length, but the CoV genome is the largest known for RNA viruses, being roughly 30kb.
  • the genomic viral RNA is used as template to directly translate polyprotein la/lab, that encodes the non-structural proteins (nsps) to form the replication-transcription complex (RTC) in a double-membrane vesicles (DMVs) [25], A nested set of sub genomic RNAs (sgRNAs) are then synthesised by RTC in a manner of discontinuous transcription [26], The sub genomic messenger RNAs (mRNAs) possess common 5'-leader and 3'-terminal sequences. Transcription termination and subsequent acquisition of a leader RNA occurs at transcription regulatory sequences, located between open reading frames (ORFs).
  • ORFs open reading frames
  • the genome and sub genomes of a typical CoV contain at least six ORFs.
  • the first ORFs (ORFla/b), about two-thirds of the whole genome length, encodes the 16 nsps (nspl-16), the other ORFs of the genome near the 3'-terminus encodes at least four main structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins.
  • S spike
  • M membrane
  • E envelope
  • N nucleocapsid
  • different CoVs encode additional special structural and accessory proteins, such as HE protein, 3a/b protein, and 4a/b protein.
  • SARS-Cov-2 is transmitted primarily via respiratory droplets; the transmission rate for SARS-Cov-2 seems to be higher when compared with both SARS-CoV and MERS.
  • the infection is asymptomatic, and these individuals are thought to be potential sources of SARS-CoV-2 infection [29], causing the rapid spread of SARS-CoV-2.
  • pneumonia appears to be the most common manifestation, other symptoms also include fever, cough, shortness of breath, and bilateral infiltrates are visible on chest imaging [30],
  • the median incubation period is approximately 4-5 days but it can be 14 days before symptoms appear [31-34], with 97.5% of symptomatic patients developing symptoms within 11.5 days [32], The viral load peaks within 5-6 days of symptoms appearing; this is significantly earlier compared to the SARS virus which peaks at 10 days after symptoms develop [35- 38], In those patients that develop acute respiratory distress syndrome, this occurs around 8-9 days after symptoms start [30, 39], SARS-CoV-2 causes an aggressive inflammatory response causing damage to the airways [40], the severity of disease is not only due to the viral infection but the host's immune response. The most common cause of death is respiratory failure (70% cases); in addition the release of cytokines induces a cytokine storm effect causing secondary infections [41] and sepsis, leading to multi organ failure and death.
  • the first step in infection is the virus binding to a host cell through its target receptor.
  • SARS-CoV-2 uses a densely glycosylated spike (S) protein to gain entry into the host cells.
  • the S protein is a trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes a structural rearrangement to fuse the viral membrane with the host cell membrane [42, 43]; this process is triggered when the SI subunit binds to a host cell receptor.
  • the SARS-CoV-2 virus binds to the angiotensin converting enzyme 2 (ACE2) receptor [24]; the serine protease TMPRSS2 is also reported to play an important role in host cell entry [44],
  • the virus targets the airway epithelial cells, alveolar epithelial cells, vascular endothelial cells and macrophages in the lung, all of which express ACE2 [24, 45],
  • the SI subunit consists of an amino- terminal domain and a receptor-binding domain (RBD).
  • the RBD binds to ACE2 triggering endocytosis of the SARS-CoV-2 virion and exposes it to endosomal proteases [46],
  • the S2 subunit consists of a fusion peptide (FP) region and two heptad repeat regions: HR1 and HR2 [47, 48], Within the endosome, the SI subunit is cleaved away, exposing the fusion peptide, which inserts itself into the host membrane. The S2 region then folds in on itself to bring the HR1 and HR2 regions together. This leads to membrane fusion and releases the viral package into the host cytoplasm.
  • FP fusion peptide
  • SARS-CoV-2 virus Once the SARS-CoV-2 virus has gained entry into the host cell, four structural proteins are then required for virus assembly, the S, M, E, and N proteins.
  • Homotrimers of S proteins make up the spikes on the viral surface and they are responsible for attachment to host receptors [49, 50]
  • M protein shapes the virions and binds to the N protein [51, 52]
  • the E protein plays a role in virus assembly and subsequent release and is important in disease pathology [53, 54],
  • the N protein contains two domains that can each bind virus RNA genome via different mechanisms. It is reported that N protein can bind to nsp3 protein to help tether the genome to the replication-transcription complex (RTC), and package the encapsulated genome into virions [20, 55, 56],
  • the RBDs of SARS-CoV and SARS-CoV-2 have 72% homology in their amino acid sequences and a highly similar tertiary protein structure. Computational modelling and biophysical measurements indicate that the SARS-CoV-2 RBD binds to ACE2 with higher affinity when compared to RBD from SARS-CoV [57, 58], The SARS-CoV-2 S protein also contains a furin-like cleavage site, similar to MERS-CoV and human coronavirus OC43, but which is not found in SARS-CoV [59], These characteristics are likely to contribute to the increased infectivity observed with the SARS-CoV-2 and have ultimately helped the virus spread.
  • cytokine storm is thought to be due to a dysfunctional immune response and a subsequent cytokine storm effect with high levels of pro-inflammatory cytokines including IL-2, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP-la and TN Fa [30, 40, 62], likely mediated by macrophages and elevated levels of inflammatory monocytes.
  • This cytokine storm is responsible for the majority of associated severe pathologies although it is unclear to date if persistence of viral infection is required to drive the ongoing immune mediated damage.
  • SARS-CoV and MERS are both known to interfere with the Type I and type III IFN response to viral infection which in turn leads to an increase in inflammatory neutrophils and macrophages to the site of infection [63, 64], It is highly likely that SARS-CoV-2 also possesses similar mechanisms of immune subversion.
  • T-cell mediated immune response is vital for the control of viral infections [68]
  • the T lymphocyte decrease associated with severe disease is also correlated with a reduction in the polyfunctionality of CD4 T-cells, specifically those making I FNy [69]
  • a suboptimal T-cell response combined with persistent antigenic activation can lead to a functionally exhausted state [70]
  • SARS- CoV-2 patients show a correlation of disease severity with increased T cell exhaustion, reduced functional diversity [71] and decreased activation [72] suggesting that an impaired T cell response may in part be responsible for the disease progression and severity.
  • SN11 which expresses the N protein fused to modified Fc gave significantly better T-cell responses to N protein and also gave superior responses to RBD, SI and peptide RBD 417-425 than a similar construct expressing the same RBD construct but N-Fc. This suggests that the modified N-Fc is acting like an adjuvant and activating the APCs to also enhance the T-cell response to other antigens.
  • neutralising antibodies Although non-neutralising antibodies can mediate in vivo protection, neutralising antibodies have received most attention [98], For instance, polyclonal IgG concentrated from the serum of vaccinated and challenged non-human primates (NHPs) was able to protect naive NHPs against Ebola virus (EV) challenge and early studies indicated that development of an antibody response was associated with survival from EV disease [99, 100], culminating in a cocktail of three monoclonal antibodies being developed against Ebola by Regeneron. During the SARS/MERS virus life cycle, neutralising antibodies inhibit viral docking (RBD-targeting), membrane fusion (S-protein-targeting) or egress (M-protein targeting).
  • RBD-targeting viral docking
  • S-protein-targeting membrane fusion
  • M-protein targeting egress
  • the surface S glycoprotein which is critical for virus entry through engaging the host receptor and mediating virus-host membrane fusion, is the major antigen of coronaviruses.
  • Other structural proteins include the E, M and N proteins, the latter being produced first and more abundantly than the others.
  • ADE extrinsic antibody-dependent enhancement
  • flaviviruses including Zika and Dengue virus as well as respiratory syncytial virus (RSV) [106]
  • RSV respiratory syncytial virus
  • the process appears to be governed by low quality and/or low quantity nonneutralising antibodies binding to virus particles through their Fab domains, with their Fc domain engaging Fc receptors (FcRs) expressed on monocytes or macrophages, thereby facilitating viral entry and infection.
  • FcRs Fc receptors
  • ADE is mediated by the engagement of Fc receptors (FcRs) expressed on different immune cells, including monocytes, macrophages and B-cells, enabling viral cell entry in the absence of their canonical receptor or endosomal pH or proteases.
  • FcRs Fc receptors
  • Vaccines have been produced against several diseases caused by coronaviruses for animal use, including for infectious bronchitis virus in birds, canine coronavirus and feline coronavirus (FCoV) [108], However, the possibility of vaccine-enhanced disease (VED) hinders development of vaccines against respiratory viruses, including FCoV.
  • FCoV feline coronavirus
  • COVID-19 vaccines currently being developed focus on subunit vaccines that predominantly encode the S protein and stimulate VNAbs and T cell responses; it remains to be determined if they stimulate durable memory responses or avoid immune pathology.
  • potent anti-viral T cell responses includes high functional avidity combined with polyfunctionality.
  • cytokines IL-12 and IL-15 [112, 113]
  • CD8aP expression [114-116]
  • TCR affinity [117]
  • co-stimulatory molecules expressed by antigen presenting cells [112, 118]
  • maturation state of DCs The challenge is therefore to find a vaccine approach which mimics these conditions.
  • Effective vaccine strategies must demonstrate: (i) the ability to protect against heterologous viral variants that arise during independent emergence events - of note, many S-targeted antibodies have significantly reduced neutralisation titres against heterologous spike glycoproteins; (ii) the ability to elicit robust immune responses in elderly populations that are difficult to immunise and at increased risk for SARS-CoV2-induced morbidity and mortality; and (iii) avoidance of adverse vaccine outcomes, such as the vaccine-induced immune pathology that has been demonstrated following vaccination with the SARS N protein [120], Strategies are generally directed at eliciting neutralising antibodies, a proven correlate with in vivo protection; however this can come at a cost of only covering a narrow range of virus reactivity (i.e. not broadly neutralising).
  • Antibodies that bind more conserved parts of the virus tend to be less neutralising (in in vitro assays), relying instead on FcR engagement for affording in vivo protection, but could cover a broader range of protection.
  • the risk of ADE argues against the use of the full S protein, in spite of it carrying more antigenic determinants, compared to the RBD [107], Structurally, the SARS-CoV-2 RBD, like that of SARS-CoV- 1, is exposed in both known states of the S protein trimer, namely a closed state where each RBD contacts symmetrically its analogues on the other protomer and an open state in which at least one RBD domain is extended to contact ACE2.
  • the RBD is also easier to produce and generates higher levels of neutralising antibodies, a large proportion of which were directed against conformational epitopes and not necessarily associated with the ACE2 binding site [104, 105],
  • Certain polypeptides, nucleic acids and vectors in accordance with the invention aim to induce high affinity antibodies to the RBD as it has been shown that higher affinity antibodies with stronger neutralising ability carry reduced risk of ADE [107],
  • the N protein is the earliest protein expressed and is more abundant compared to the S or M protein [89], Combined with its more conserved nature and thus greater likelihood for heterologous protection, this also makes a valid target for the present invention.
  • the present invention can target both a virus N protein and the key RBD of the S protein to generate CD8 T cells, CD4 T cells and VNAbs.
  • the S protein is presented as a trimer in its interaction with ACE2 so to optimise eliciting antibodies that have the neutralising phenotype.
  • the inventors have multimerised the RBD, and this may be used in the vaccine for the prevention or treatment of SARS- CoV-2.
  • One polypeptide of the present invention comprises the N protein or an immunogenic fragment thereof fused to the modified human lgG3 Fc region.
  • the N protein or immunogenic fragment thereof may comprise amino acids 2-419 or 138-146.
  • the modified human lgG3 Fc region may comprise the Hinge-CH2-CH3 regions having the murine lgG3 modifications described herein.
  • Figure 12b This polypeptide may be in combination with a second polypeptide comprising the RBD or an immunogenic fragment thereof.
  • the RBD may comprise amino acids 319- 541 or 330-525 (Accession Number YP_009724390).
  • the RBD may (a) alone, (b) attached to a trimerization domain, such as fibritin trimer fold on or disulphide bridge motif, for example via a glycine serine linker or (c) fused in frame with (i) the Hinge-CH2-CH3 domain of the HuIgGl constant domain (Accession Number P01857) or (ii) the variant Hinge-CH2-CH3iVl in accordance with the invention. Examples of these are shown in Table 4 herein.
  • infectious diseases such as viral infections (as described above) and bacterial infections
  • the invention can be used to target tumour antigens. Examples of such antigens are set out in Table 3 herein.
  • Tumours accumulate mutations that drive growth and metastases. These mutations represent unique epitopes that avoid thymic selection. They are termed “neo-epitopes" and are specific to individual tumours and are not found on normal tissues [121], Lennerz et al. identified in a mixed lymphocyte-tumour cell culture from one patient with long-term survival from melanoma responses to eight antigens, five of which were neo-antigens [122], This is an early study that showed that neo-antigens are associated with responses in long-term survivors. This has led researchers to develop personalised vaccines against identified neo-epitopes.
  • neo-epitopes identified in these studies were recognised by both CD8 and CD4 T cells suggesting an important role for CD4 T cell responses in addition to CD8 responses in humans.
  • T cells specific for self-antigens are routinely deleted in the thymus during development leaving a low avidity repertoire. Therefore, antigens that show limited normal expression are likely to act as better targets since they may not have been subject to the same degree of tolerance.
  • the detection of T cells specific to self-antigens in regressing cancer patients suggests that thymic tolerance is not always complete.
  • TAA Tumour associated antigens
  • T cells require the processing and presentation of antigen by professional antigen presenting cells (APCs) such as dendritic cells (DCs) along with appropriate activating costimulatory signals.
  • APCs professional antigen presenting cells
  • DCs dendritic cells
  • Activating costimulatory signals include those provided by TLR ligands [reviewed in 135], Preclinical studies examining linkage of the peptide vaccine directly to TLR ligands are beginning to show promise.
  • T cell functional avidity is a better indicator of clinical response [141-145]
  • the term 1 functional avidity is often confused with affinity.
  • Affinity is most often classified as a measure of the strength of binding of the peptide MHC molecule to the T cell receptor (TCR) whereas functional avidity is a measure of the combination of stimulation via TCR, co-stimulatory molecules, adhesion molecules and cytokines and is indicative of the overall strength of interaction between T cell and target and its functional outcome [146],
  • TCL cytotoxic T lymphocytes
  • Peptide vaccines encoding tumour epitopes have shown promise in animal models in early studies, stimulating specific T cell responses and tumour therapy in mice. Translation of these peptide vaccines into the clinic has been less successful with responses being short lived and minimal clinical efficacy. Early vaccines concentrated on the stimulation of CD8 T cell responses with short ( ⁇ 15 amino acids) peptides.
  • Synthetic peptides have also been used as part of DC based vaccines. Many studies have been performed where DCs cultured in vitro have been pulsed with peptides, proteins or tumour lysates. These have shown stimulation of efficient immune responses in preclinical studies [reviewed in 154], Despite stimulating immune responses, DC vaccines have shown limited efficacy in the clinic. Sipuleucel-T (Provenge®), the only approved therapeutic autologous cell based vaccine to date, has shown a modest survival benefit of 3 months but the cost and time of production have severely limited its use [155], This is the major limiting factor of most DC and autologous cell-based vaccines.
  • the nucleic acid of the invention may be DNA, cDNA, or RNA such as mRNA, obtained by cloning or produced wholly or partly by chemical synthesis.
  • the nucleic acid is preferably in a form capable of being expressed in the subject to be treated.
  • the nucleic acid of the present invention may be recombinant or provided as an isolate, in isolated and/or purified form. It may be free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence(s) for expression.
  • nucleic acid according to the invention includes RNA, reference to the sequences shown herein should be construed as reference to the RNA equivalent, with U substituted forT.
  • Nucleic acids of the present invention can be readily prepared by the skilled person, for example using the information and references contained herein and techniques known in the art (for example, see Sambrook et al., (1989) [157], and Ausubel et al., (1992) [158], given the nucleic acid sequences and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences.
  • PCR polymerase chain reaction
  • DNA encoding the polypeptide may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preferences in the host cells used to express the nucleic acid.
  • the sequences can be incorporated into a vector having one or more control sequences operably linked to the nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • polypeptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the polypeptide is produced and recovering the polypeptide from the host cells or the surrounding medium.
  • Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as insect cells, and animal cells, for example, COS, CHO cells, Bowes Melanoma and other suitable human cells.
  • the present invention relates to nucleic acid(s) encoding the heavy and light chains of an antibody, the respective nucleic acids may be present in the same expression vector, driven by the same or different promoters, or in separate expression vectors.
  • the nucleic acids of the present invention may be used to stimulate an immune response against the at least one heterologous antigen in a patient such as a mammal, including human. Helper and/or cytotoxic T cell responses may be stimulated.
  • the T cell response against a particular epitope obtained by the present invention may have a higher avidity than that obtained by immunisation with the same epitope as a simple peptide, or by immunisation with the same epitope encoded within an antigen either as a peptide or a nucleic acid.
  • the nucleic acids of the invention may be administered as a combination therapy, i.e. a nucleic acid encoding the light chain and nucleic acid encoding the heavy chain.
  • the nucleic acid may be administered intravenously, intradermally, intramuscularly, orally or by other routes. Intradermal or intramuscular administration is preferred because these tissues contain dendritic cells.
  • a further aspect of the invention provides a vector comprising the nucleic acid of the invention.
  • Vectors may be used for expression of the nucleic acid in order to obtain the polypeptide of the invention, or they may be used as a treatment (e.g. vaccine) in and of themselves.
  • Exemplary vectors include iSCI Blplus (see Figure 31), iSCI B2 (see Figure 33), SN15 (see Figure 27) and SN17 (see Figure 48).
  • the vector of the invention may comprise the nucleotide sequences provided in:
  • SEQ ID NO: 12 and SEQ ID NO: 13 are the nucleotide sequences of the whole iSCIBlplus heavy and light chain expression cassettes respectively, both sequences including a CMV promoter and BGH polyA signal.
  • SEQ ID NO: 14 and SEQ ID NO: 15 are the nucleotide sequences of the whole iSCI B2 heavy and light chain expression cassettes respectively, both sequences including a CMV promoter and BGH polyA signal.
  • the vector of the invention may comprise the nucleotide sequences provided in SEQ ID NO: 16 and SEQ. ID NO: 17.
  • SEQ ID NO: 16 and SEQ ID NO: 17 are the nucleotide sequences of the whole SN15 N protein-Fc and S protein expression cassettes respectively, both sequences including a CMV promoter and BGH polyA signal.
  • the vector of the invention may comprise the nucleotide sequences provided in SEQ ID NO: 18 and SEQ ID NO: 19.
  • SEQ ID NO: 18 and SEQ ID NO: 19 are the nucleotide sequences of the whole SN17 N protein-Fc and S protein expression cassettes respectively, both sequences including a CMV promoter and BGH polyA signal.
  • the vector of the invention may comprise the nucleotide sequence provided in SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
  • the vector of the invention consists of the nucleotide sequence provided in SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
  • SEQ ID NO: 20 is the whole iSCIBlplus nucleotide sequence in plasmid format.
  • SEQ ID NO: 21 is the whole iSCIBlplus nucleotide sequence in doggybone (dbDNA) format.
  • SEQ ID NO: 22 is the whole iSCI B2 nucleotide sequence in plasmid format.
  • the vector of the invention may comprise the nucleotide sequence provided in SEQ ID NO: 23.
  • the vector of the invention consists of the nucleotide sequence provided in SEQ ID NO: 23.
  • SEQ ID NO: 23 is the whole SN15 vector nucleotide sequence, in plasmid format.
  • the vector of the invention may comprise the nucleotide sequence provided in SEQ ID NO: 24 or SEQ ID NO: 25.
  • the vector of the invention consists of the nucleotide sequence provided in SEQ ID NO: 24 or SEQ ID NO: 25.
  • SEQ ID NO: 24 is the whole SN17 vector nucleotide sequence, in plasmid format.
  • SEQ ID NO: 25 is the whole SN17 vector nucleotide sequence, in doggybone (dbDNA) format.
  • the vector of the invention may be DNA. Plasmid DNA vaccines offer a number of advantages over other vaccine modalities as they have intrinsic adjuvant activity resulting in recruitment of large numbers of inflammatory cells to the site of immunisation.
  • DNA vaccine-induced immunity The mechanisms underlying DNA vaccine-induced immunity are complex and have yet to be fully elucidated but are thought to involve promiscuous and discriminative DNA sensors expressed by APCs (Table 1). CpG motifs signal through TLR9 to promote the activation and maturation of DCs [159], Interestingly, DNA vaccine activity was still observed in TLR9 knockout mice implicating additional endosomal and cytosolic DNA sensors that mediate adjuvant activity [160], DNA sensors such as TBK-1 and STING activate TLR-lndependent pathways and induce type I interferons [161], More recently the helicase DDX41 was identified as a new DNA sensor in myeloid dendritic cells [162], In addition RIG-1 can also stimulate type I IFNs by sensing cytosolic DNA in association with RNA polymerase III [163], DNA- dependent activator of IFN regulatory factors (DAI/DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immunity [164],
  • HMGB-1 has emerged as promiscuous sensor for nucleic acid mediated induction of innate immune responses [166], HMGB-1 (a chromatin binding protein), has a range of functions depending on its subcellular and extracellular localisation, redox state, and interaction with other cell surface receptors. As an intracellular complex, HMGB-1 associated nucleic acids stimulate through TLR and cytosolic mediated sensors, type I IFNs, activate pro-inflammatory cytokines and induce the inflammasome (though AIM2) (reviewed in [160]). Extracellular release of HMGB-1 has a range of consequences, including sustaining tumour cell autophagy (by competing with bcl-2 for beclin-1 binding, as well as recruitment and activation of immune cells [167]).
  • HMGB-l contains three cysteines at positions C23, C45 and C106 that can be modified.
  • the reduced all-thiol form of HMGB-l is a chemoattractant that mediates leukocyte recruitment.
  • the disulphide form has cytokine (but not chemokine) activity.
  • the fully oxidised form of HMGB-l induced by reactive oxygen species is inactive.
  • HMGB-l also forms complexes with cytokine and other immune receptors including RAGE, TLR4, TLR2, CD24, TIM-3, thrombospondin and TREM1 [168, 169], HMGB- 1/DNA complexes bind to RAGE and induce a switch from apoptosis to autophagy [169], HMGB- 1/CXCL12 binds to CXCR4 mediating recruitment of inflammatory cells [170],
  • the vector of the invention is a DNA plasmid or doggybone (dbDNA) vector.
  • dbDNA vectors DNA plasmid or doggybone vectors.
  • dbDNA vectors, and methods of production are described in WO 2010/086626.
  • the vector of the present invention may be RNA.
  • RNA vaccines There are two categories of mRNA vaccines, those using non-replicating mRNA and those using self-replicating RNA. The invention contemplates both.
  • the non-replicating mRNA vaccines contain only the transcript for the antigen of interest, whereas the self-replicating RNA vaccines in addition to the antigen of interest also include transcripts for the RNA replication machinery required for mRNA amplification.
  • the self-replicating RNA vaccines induce the production of a large amount of antigen from only a small dose; this has the advantage that development and manufacturing of the vaccine is less complicated and much cheaper compared to other platforms.
  • RNA vaccines have a short half-life and therefore do not sustain the production of the antigen giving short lived protection.
  • the mRNA For both non-replicating and selfreplicating mRNA vaccines, the mRNA must be formulated to prevent degradation or be enclosed within a carrier which protects the mRNA from degradation by nucleases prior to uptake by host cells.
  • a carrier which protects the mRNA from degradation by nucleases prior to uptake by host cells.
  • a number of different carriers have been successfully used [171-174], these are mainly based on lipid-nanoparticles that encapsulate the mRNA and facilitate cellular uptake.
  • the present invention contemplates the use of these mRNA vaccines.
  • the present invention provides
  • a vaccine comprising polypeptide, nucleic acid and/or vector of the present invention, optionally in combination with an adjuvant,
  • a method for treating cancer or an infectious disease comprising administering to a subject in need of such treatment a polypeptide, nucleic acid, vector and/or vaccine of the present invention.
  • the term "treatment" includes any regime that can benefit a human or non-human animal.
  • the treatment may be of an inherited or acquired disease.
  • the treatment is of a condition/disorder associated with cell proliferation such as cancer or infectious disease.
  • types of cancer that can be treated with the nucleic acid include any solid tumour, colorectal cancer, lung, breast, gastric, ovarian, uterine, liver, kidney, pancreatic, melanoma, bladder, head and neck, brain, oesophageal, pancreatic, and bone tumours, as well as soft tissue cancers, and leukaemia's.
  • infectious diseases that can be treated with the invention include infection with bacteria or viruses, such as coronaviruses, HIV, Hepatitis C, or any infection that requires T cell immunity for clearance and neutralising mAbs to prevent re-infection.
  • the present invention provides a nucleic acid, peptide, vector and/or vaccine described herein for use in the prevention or treatment of cancer in a subject, optionally wherein the cancer is melanoma.
  • the present invention provides a nucleic acid, peptide, vector and/or vaccine described herein for use in the prevention or treatment of an infectious disease in a subject, optionally wherein the infectious disease is COVID-19.
  • Two or more different nucleic acids vectors peptides and/or vaccines may be administered to the subject.
  • both of the following nucleic acids are administered to the subject:
  • nucleic acid encoding a polypeptide comprising the amino acid sequence provided in SEQ. ID NO: 6, in combination with a second nucleic acid encoding a receptor binding domain comprising the amino acid sequence provided in SEQ ID NO: 8;
  • nucleic acid, polypeptide and/or vector may be employed in combination with a pharmaceutically acceptable carrier or carriers.
  • carriers may include, but are not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, ethanol and combinations thereof.
  • nucleic acids, polypeptides and/or vectors useful in the invention can be formulated in pharmaceutical compositions.
  • These compositions may comprise, in addition to one or more of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. intradermal, oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • the formulation is preferably nucleic acid as a stable dry powder precipitated onto the surface of microscopic gold particles and suitable for injection via a gene gun or a solution of DNA mixed with GET peptides.
  • the formulation may be suitable for intradermal or intramuscular administration using electroporation.
  • the formulation may be suitable for administration using needle-free injection.
  • compositions comprising, or for the delivery of, nucleic acids are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • the nucleic acids of the invention are particularly relevant to the treatment of existing cancer and in the prevention of the recurrence of cancer after initial treatment or surgery. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16 th edition, Oslo, A. (ed), 1980.
  • the nucleic acid of the invention stimulates helper and/or cytotoxic T cells that can significantly kill virally infected cells or generate VNAbs to prevent viral entry when administered to a human in an effective amount.
  • the optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration. For example, a dose of l-1000pg of DNA is sufficient to stimulate both helper and cytotoxic T cell responses.
  • a composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • Other cancer treatments include other monoclonal antibodies, other chemotherapeutic agents, other radiotherapy techniques or other immunotherapy known in the art.
  • the dose of nucleic acid will be dependent upon the properties of the agent employed, e.g. its binding activity and in vivo plasma half-life, the concentration of the polypeptide in the formulation, the administration route, the site and rate of dosage, the clinical tolerance of the patient involved, the pathological condition afflicting the patient and the like, as is well within the skill of the physician.
  • doses of 200pg of nucleic acid per patient per administration are preferred, although dosages may range from about 10pg to 8mg per dose. Different dosages are utilised during a series of sequential inoculations; the practitioner may administer an initial inoculation and then boost with relatively smaller doses of nucleic acid.
  • a further aspect of the present invention provides a host cell containing a nucleic acid as disclosed herein.
  • the nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome in accordance with standard techniques.
  • the nucleic acid may be on an extra- chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.
  • a still further aspect provides a method, which comprises introducing the nucleic acid of the invention into a host cell.
  • the introduction which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation", may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • direct injection of the nucleic acid could be employed.
  • Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide (or peptide) is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.
  • a polypeptide or peptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).
  • the pharmaceutical composition may comprise, in addition to active ingredient, pharmaceutically acceptable excipient, diluent, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • pharmaceutically acceptable excipient diluent, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g., intradermal or intramuscular.
  • injections will be the primary route for therapeutic administration of the compositions although delivery through a catheter or other surgical tubing is also used.
  • Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal and intramuscular administration.
  • Liquid formulations may be utilised after reconstitution from powder formulations.
  • Preferred routes of administration are intradermal or intramuscular administration.
  • the nucleic acid, vector, peptide and/or vaccine of the invention may be administered to the subject using needle-free injection.
  • needle-free injectors also known as “jet injectors"
  • jet injectors use a narrow, high-pressure stream of liquid that penetrates the outermost layer of the skin (stratum corneum) to deliver a composition to underlying tissues of the epidermis or dermis (i.e., intradermal injection), fat (i.e., subcutaneous injection), or muscle (i.e., intramuscular injection).
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may comprise a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Where the formulation is a liquid it may be for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised powder.
  • composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood.
  • sustained release carriers include semi-permeable polymer matrices in the form of shared articles, e.g., suppositories or microcapsules.
  • Implantable or microcapsular sustained release matrices include polylactides (US Patent No. 3, 773, 919; EP-A-0058481) copolymers of L-glutamic acid and gamma ethyl-L-glutamate [43], poly (2-hydroxyethyl-methacrylate).
  • Liposomes containing the polypeptides are prepared by well-known methods: [175, 176]; EP-A-0052522; EP-A-0036676; EP-A-0088046; EP-A- 0143949; EP-A-0142541; JP-A-83-11808; US Patent Nos 4,485,045 and 4,544,545.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. % cholesterol, the selected proportion being adjusted for the optimal rate of the polypeptide leakage.
  • the composition may be administered in a localised manner to a tumour site or other desired site or may be delivered in a manner in which it targets tumour or other cells.
  • polypeptides of the present invention may be generated wholly or partly by chemical synthesis.
  • the polypeptide can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, J.M. Stewart and J.D. Young, (1984) [177], in M. Bodanzsky and A.
  • nucleic acid includes DNA and RNA.
  • the skilled person will be able to determine substitutions, deletions and/or additions to such nucleic acids which will still provide the polypeptide of the present invention.
  • the present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one nucleic acid as described above.
  • the present invention also provides a recombinant host cell which comprises one or more constructs as above.
  • a nucleic acid encoding the polypeptide of the invention forms an aspect of the present invention, as does a method of production of the polypeptide which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression the polypeptide may be isolated and/or purified using any suitable technique, then used as appropriate.
  • Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others.
  • a common, preferred bacterial host is E. coli.
  • the expression of polypeptides in prokaryotic cells such as E. coli is well established in the art. For a review see for example [179], Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of the polypeptides of the invention, see for recent review, for example [180, 181],
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g., 'phage, or phagemid, as appropriate.
  • plasmids viral e.g., 'phage, or phagemid, as appropriate.
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Ausubel et al. , 1992 [182],
  • a further aspect of the present invention provides a host cell containing nucleic acid in accordance with the invention.
  • a still further aspect provides a method comprising introducing such nucleic acid into a host cell.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • the introduction may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene.
  • the nucleic acid of the invention is integrated into the genome (e.g., chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.
  • the present invention also provides a method which comprises using a construct as stated above in an expression system in order to express the polypeptide as above.
  • the Fragment crystallizable region is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.
  • the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains.
  • the Fc regions of IgGs comprise a highly conserved N-glycosylation site.
  • the Fc region of an IgG consists of a paired set of antibody HC domains, each of which has a CH2 fused to a CH3, which form a structure of about 50 kDa.
  • the name "Fragment, crystallizable” (Fc) comes from the fact that after cleavage of serum-derived myeloma IgG fractions with papain, the only fragment that could be crystallized was the paired CH2-CH3 fragment.
  • the two CH3 domains bind each other tightly, whereas the two CH2 domains have no direct protein-protein contact with one another.
  • An oligosaccharide is bound to asparagine-297 (N297) within each of the two CH2 domains, filling part of the space between the two CH2s.
  • hydrogen bonding has been observed between the two carbohydrate chains, directly and through bridging water molecules.
  • the antibody appears to be a highly segmented molecule, it has been demonstrated that the structure of the Fc can impact the binding of the antigen-binding fragments (Fabs) to the targeted antigens and, similarly, that the content of the variable chain in the FAbs can impact binding of the Fc to various receptors.
  • Fabs antigen-binding fragments
  • Recently circular dichroism studies have confirmed significant structural coupling between the FAb arms and the Fc of the IgG.
  • the IgG molecule is a highly complex molecule in which the different domains significantly interact, even at long distances.
  • Avidity refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, such as between a protein receptor and its ligand, and can also be referred to as 'functional affinity'. As such, avidity is distinct from intrinsic affinity, which describes the strength of a single interaction. However, because individual binding events increase the likelihood of other interactions to occur (i.e. increase the local concentration of each binding partner in proximity to the binding site), avidity should not be thought of as the mere sum of constituent affinities but as the combined effect of all affinities participating in the biomolecular interaction. The utility of the distinction between "intrinsic affinity” and “functional affinity” arises from the different emphasis involved in each term.
  • the former is most useful when the structural relationship between the antibody combining site and the complementary region of the ligand is under scrutiny or when kinetic mechanisms of the specific interaction are under investigation.
  • the latter is particularly significant when the quantitative measurement of the enhancement of affinity is being examined, as in the present invention.
  • the present invention also extends to variants of any peptide sequences disclosed herein.
  • variant relates to proteins that have a similar amino acid sequence and/or that retain the same function.
  • variant encompasses proteins or polypeptides which include one or more amino acid additions, deletions, substitutions or the like.
  • An example of a variant of the present invention is a protein comprising a peptide as defined below, apart from the substitution of one or more amino acids with one or more other amino acids. Amino acid substitutions may be made to, for example, reduce or eliminate liabilities in the amino acid sequences.
  • amino acids have similar properties.
  • One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance.
  • amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • amino acids having aliphatic side chains amino acids having aliphatic side chains.
  • glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic).
  • amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Substitutions of this nature are often referred to as “conservative" or “semi- conservative" amino acid substitutions.
  • glycine G or Gly
  • alanine A or Ala
  • valine V or Vai
  • leucine L or Leu
  • isoleucine I or He
  • proline P or Pro
  • phenylalanine F or Phe
  • tyrosine Y or Tyr
  • tryptophan W or Trp
  • lysine K or Lys
  • arginine R or Arg
  • histidine H or His
  • aspartic acid D or Asp
  • glutamic acid E or Glu
  • asparagine N or Asn
  • glutamine Q.
  • a residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where a residue may be glutamic acid or glutamine, the symbols Glx or Z may be used. References to aspartic acid include aspartate, and glutamic acid include glutamate, unless the context specifies otherwise.
  • Amino acid deletions or insertions can also be made relative to the amino acid sequence for the fusion protein referred to below.
  • amino acids which do not have a substantial effect on the activity of the polypeptide, or at least which do not eliminate such activity can be deleted.
  • Such deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.
  • amino acids can be exchange for each other for conservative amino acid substitutions:
  • references to “conservative” amino acid substitutions refer to amino acid substitutions in which one or more of the amino acids in the sequence of the antibody (e.g. in the CDRs or in the VH or VL sequences) is substituted with another amino acid in the same class as indicated above.
  • Conservative amino acid substitutions may be preferred in the CDR regions to minimise adverse effects on the function of the antibody.
  • conservative amino acid substitutions may also occur in the framework regions.
  • Amino acid changes relative to the sequence given below can be made using any suitable technique e.g. by using site-directed mutagenesis or solid-state synthesis.
  • amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids, although naturally occurring amino acids may be preferred. Whether or not natural or synthetic amino acids are used, it may be preferred that only L- amino acids are present.
  • the invention also provides
  • a nucleic acid vaccine whereby the antigen is a tumour specific or tumour associated antigen.
  • a nucleic acid vaccine whereby the antigen is a viral or bacterial antigen.
  • a nucleic acid vaccine whereby the antigens are two viral proteins that generate both T cell responses and VNAbs.
  • a nucleic acid vaccine whereby the antigens are two viral proteins to generate CD8 T cells, CD4 T cells and VNAbs.
  • a nucleic acid vaccine whereby the antigens are a virus nucleocapsid protein and the key receptor-binding domain of the spike protein to generate both T cell responses and VNAbs.
  • a nucleic acid vaccine whereby the antigens are a virus nucleocapsid protein and the key receptor-binding domain of the spike protein to generate CD8 T cells, CD4 T cells and VNAbs.
  • a nucleic acid vaccine whereby the antigens are a coronavirus nucleocapsid protein and the key receptor-binding domain of the spike protein to generate both T cell responses and VNAbs.
  • a nucleic acid vaccine whereby the antigens are both coronavirus nucleocapsid protein and the key receptor-binding domain of the spike protein to generate CD8 T cells, CD4 T cells and VNAbs.
  • a nucleic acid vaccine whereby the antigens are both the SARS-CoV-2 coronavirus nucleocapsid protein and the key receptor-binding domain of the spike protein to generate both T cell responses and VNAbs.
  • a nucleic acid vaccine whereby the antigens are both SARS-CoV-2 coronavirus nucleocapsid protein and the key receptor-binding domain of the spike protein to generate CD8 T cells, CD4 T cells and VNAbs.
  • a nucleic acid vaccine encoding the COVID-19 spike receptor binding domain aa 319-541 with a human heavy chain leader sequence and the nucleocapsid protein linked to the hinge and CH2 and CH3 domains of human IgGl Fc with a human heavy chain leader sequence.
  • a nucleic acid vaccine encoding the COVID-19 spike receptor binding domain aa 319-541 linked to fibritin with a human heavy chain leader sequence and the nucleocapsid protein linked to the hinge and CH2 and CH3 domains of human IgGl Fc with a human heavy leader sequence.
  • a nucleic acid vaccine encoding the COVID-19 spike receptor binding domain aa 319-541 linked to the hinge and CH2 and CH3 domains of human IgGl Fc with a human heavy chain leader sequence and the nucleocapsid protein with a human heavy chain leader sequence.
  • a nucleic acid vaccine encoding the COVID-19 spike receptor binding domain aa 319-541 with a human heavy chain leader sequence and the nucleocapsid protein with a human heavy chain leader sequence.
  • a nucleic acid vaccine encoding the COVID-19 spike receptor binding domain aa 330-525 with a GS linker and fibritin with a human heavy chain leader sequence and the nucleocapsid protein linked to the hinge and CH2 and CH3 domains of human IgGl Fc with a human heavy chain leader sequence.
  • a nucleic acid vaccine encoding the COVID-19 spike receptor binding domain aa 330-525 with a GS linker and additional cysteine residues with a human heavy chain leader sequence and the nucleocapsid protein linked to the hinge and CH2 and CH3 domains of human IgGl Fc with a human heavy chain leader sequence.
  • a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the modified IgGl antibody or antigen binding fragment thereof has enhanced functional affinity when compared to a corresponding IgGl antibody or antigen binding fragment thereof comprising wildtype Fc region residues.
  • a nucleic acid vaccine comprising a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the modified IgGl antibody or antigen binding fragment thereof has enhanced functional affinity when compared to a corresponding IgGl antibody or antigen binding fragment thereof comprising wildtype Fc region residues.
  • a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: Q342, P343, E345, N361, Q362, P374, D376, optionally wherein the one or more modified residues of the Fc region are selected from: Q342R, P343A, E345T, N361K, Q362K, P374S, D376A.
  • a nucleic acid vaccine comprising a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: Q.342, P343, E345, N361, Q362, P374, D376, optionally wherein the one or more modified residues of the Fc region are selected from: Q342R, P343A, E345T, N361K, Q362K, P374S, D376A.
  • a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378, optionally wherein the one or more modified residues of the Fc region are selected from: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.
  • a nucleic acid vaccine comprising a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: N286, K288, K290, Q342, P343, E345, L351, T359, N361, Q362, G371, P374, S375, D376, A378, optionally wherein the one or more modified residues of the Fc region are selected from: N286T, K288W, K290Q, Q342R, P343A, E345T, L351I, T359S, N361K, Q362K, G371N, P374S, S375E, D376A, A378S.
  • a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378, optionally wherein the one or more modified residues of the Fc region are selected from: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362
  • a nucleic acid vaccine comprising a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: N286, K288, K290, A339, Q.342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378, optionally wherein the one or more modified residues of the Fc region are selected from: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359
  • a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378, optionally wherein the one or more modified residues of the Fc region are selected from: N286T, K288W, K290Q, E294A, Y300F, V305A, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E
  • a nucleic acid vaccine comprising a modified IgGl antibody or antigen binding fragment thereof comprising one or more residues of an Fc region of an immunoglobulin and a binding region, wherein one or more residues of the Fc region are modified to the corresponding residue from a mouse lgG3 antibody and wherein the one or more residues of the Fc region are selected from: N286, K288, K290, E294, Y300, V305, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378, optionally wherein the one or more modified residues of the Fc region are selected from: N286T, K288W, K290Q., E294A, Y300F, V305A, A339P, Q.342R,
  • FIG. 1 Map of pVAXDCIB68 and cloning strategy
  • Nucleotide and amino acid sequence of the S glycoprotein and N full length chains within the expression vector pVAXDC The S chain encodes RBD amino acids 319-541 and a murine IgK leader.
  • the nucleoprotein chain encodes amino acids 2-419 fused inframe with the human IgGl hinge-CH2- CH3 along with the murine IgK leader.
  • the stop codon is depicted by an asterix. The BamHI/Xhol and Hind I ll/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • the S chain encodes RBD amino acids 319-541 linked via a glycine serine to a fibritin trimer motif.
  • the N chain encodes amino acids 2-419 fused inframe with the human igGl hinge-CH2-CH3. The stop codon is depicted by an asterix.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC Both chains contain a murine IgK leader.
  • the S chain encodes RBD amino acids 319-541 fused inframe with the human IgGl hinge-CH2-CH3.
  • the N chain encodes amino acids 2-419.
  • the stop codon is depicted by an asterix. The BamHI/Xhol and Hind I ll/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC Both chains contain a murine IgK leader.
  • the S glycoprotein chain encodes RBD amino acids 319-541 while the N chain encodes amino acids 2- 419.
  • the stop codon is depicted by an asterix. The BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC The S chain encodes RBD amino acids 319-541 and a human IgH leader.
  • the N chain encodes amino acids 2-419 fused in frame with the human IgGl hinge-CH2-CH3 along with the human IgH leader.
  • the stop codon is depicted by an asterix. The BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC Both chains contain a human IgH leader.
  • the S chain encodes RBD amino acids 319-541 linked via a glycine serine to a fibritin trimer motif.
  • the N chain encodes amino acids 2-419 fused in frame with the human IgGl hinge-CH2-CH3.
  • the stop codon is depicted by an asterix.
  • the BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC Both chains contain a human IgH leader.
  • the S chain encodes RBD amino acids 319-541 fused inframe with the human IgGl hinge-CH2-CH3.
  • the N chain encodes amino acids 2-419.
  • the stop codon is depicted by an asterix. The BamHI/Xhol and Hind I ll/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC Both chains contain a human IgH leader.
  • the S glycoprotein chain encodes RBD amino acids 319-541 while the N chain encodes amino acids 2- 419.
  • the stop codon is depicted by an asterix. The BamHI/Xhol and Hind I ll/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC Both chains contain a human IgH leader.
  • the S chain encodes RBD amino acids 330-525 attached via a longer (GGGSjaGS glycine serine linker to a fibritin trimer motif (GTGGGSG).
  • the N chain encodes amino acids 2-419 fused in frame with the human IgGl hinge-CH2-CH3.
  • the stop codon is depicted by an asterix.
  • the BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC Both chains contain a human IgH leader.
  • the S chain encodes RBD amino acids 330-525 attached via a (GGGS)a glycine serine linker to a disulphide bridge motif.
  • the N chain encodes amino acids 2-419 fused in frame with the human IgGl hinge-CH2-CH3.
  • the stop codon is depicted by an asterix.
  • the BamHI/Xhol and Hind I ll/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • the S chain encodes RBD amino acids 330-525 attached via a longer (GGGSjaGS glycine serine linker to a fibritin trimer motif (GTGGGSG).
  • the N chain encodes amino acids 2-419 fused in frame with the human IgGl hinge-CH2-CH3 iVl where murine lgG3 23 AA substitutions are highlighted in bold.
  • the stop codon is depicted by an asterix.
  • the BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Figure 13 Sequence of pVAXDCSN12; SN12 Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader.
  • the S chain encodes RBD amino acids 319-541 linked via a glycine serine to a fibritin trimer motif.
  • the N chain encodes amino acids 2-419 fused in frame with the human IgGl hinge-CH2-CH3 iVl where murine igG3 23 AA substitutions are highlighted in bold.
  • the stop codon is depicted by an asterix.
  • the BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • the S chain encodes RBD amino acids 319-541 fused in frame with the human IgGl hinge-CH2-CH3.
  • the N chain encodes amino acids 2-419 fused in frame with the human IgGl hinge-CH2-CH3 iVl where murine igG3 23 AA substitutions are highlighted in bold.
  • the stop codon is depicted by an asterix.
  • the BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Figure 15 Sequence of pVAXDCSN14; SN14
  • the S chain encodes RBD amino acids 319-541 while the N chain encodes amino acids 2-419 both fused in frame with the human IgGl hinge-CH2- CH3 iVl constant region where murine igG3 23 AA substitutions are highlighted in bold.
  • the stop codon is depicted by an asterix.
  • the BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Figure 16 RBD and N secretion levels (by sandwich ELISA) in conditioned medium six days after transient HEK293 transfections with respective SN constructs.
  • FIG. 17 HHDII mice immunised on days 1, 8 and 15 with SN8, SN9, SN10 and SN11 (A) or SN8, SN10 and SN11 (B) pDNA via gene gun.
  • FIG. 18 HHDII/DR1 mice immunised on days 1, 8 and 15 with SN8 (A), SN9 (B), SN10 (C) and SN11 (D) pDNA via gene gun.
  • Figure 19 Comparison of IFNy responses in ELISpot to N protein (A), N 138-147 peptide (B), SI protein (C) and RBD protein (D) from mice immunised with SN8, SN9 or SN11 pDNA via gene gun. Data collated from two independent studies. Responses normalised against background control. Responses displayed as average spots/million splenocytes.
  • FIG. 20 HHDII/DP4 mice immunised on days 1, 15 and 29 with SN2 (A), SN3 (B) and SN4 (C) pDNA via gene gun.
  • Figure 21 HHDII/DP4 mice immunised on days 1, 15 and 29 with SN2, SN3 and SN4 pDNA via gene gun. Splenocytes analysed at day 35 for IFNy responses to titrating amounts of SI protein by ELISpot assay. Avidity calculated as protein concentration which elicits 50% maximal response. Titration curves shown as spots/million splenocytes and responses normalised to display as a % maximal response curve.
  • FIG. 22 HHDII mice immunised on days 1, 8 and 15 with SN11 pDNA via gene gun. Splenocytes analysed at day 21 for IFNy responses to titrating amounts of RBD 417-425 peptide by ELISpot assay. Avidity calculated as protein concentration which elicits 50% maximal response. Titration curves shown as spots/million splenocytes.
  • FIG 23 HHDII mice immunised on days 1, 8 and 15 with SN10 or SN11 pDNA via gene gun. Splenocytes analysed at day 21 for IFNy responses to titrating amounts of N 138-146 peptide by ELISpot assay. Avidity calculated as protein concentration which elicits 50% maximal response. Titration curves shown as spots/million splenocytes.
  • Figure 24 HHDII/DP4 mice immunised on days 1, 15 and 29 with SN5, SN6, SN9, SN10 and SN11 (A) SN2, SN3 and SN4 (B) pDNA or on days 1, 8, 15 with SN3, SN8, SN10 and SN11 (C) pDNA via gene gun. Sera at 1/100, 1/1000 and 1/10000 dilutions analysed at day 35 (A and B) or day 21 (C) for antibody responses to SI, N and RBD proteins by ELISA.
  • Figure 25 Surrogate neutralisation assay (RBD binding inhibition assay) on sera taken at day 35 from HHDII/DP4 mice immunised with SN5, SN6, SN9, SN10 and SN11 via gene gun on days 1, 15 and 29. Sera from naive mice used as a negative control and murine SI antibody (SinoBiological) as an additional positive control.
  • RBD binding inhibition assay Surrogate neutralisation assay
  • Figure 26 Pseudovirus neutralisation assay. HHDII/DP4 mice immunised on days 1, 15 and 29 with SN5, SN6, SN9, SN10 and SN11, SN2, SN3 and SN4 pDNA via gene gun. Sera taken at day 35 was tested at 1/100 dilution for neutralisation of SARS-CoV-2 (A) or an irrelevant virus (VSV G) (B). Virus neutralisation was also analysed at different sera dilutions (C). 50% neutralisation titres (ID50) (D).
  • Figure 27 Sequence of pVAXDCSN15; SN15
  • Nucleotide and amino acid sequence of the spike and nucleoprotein full length chains within the expression vector pVAXDC Both chains contain a human IgH leader.
  • the spike chain encodes amino acids 319-541.
  • the nucleoprotein chain encodes amino acids 2-419 fused inframe with the human igGl hinge-CH2-CH3 iVl where murine igG3 23 AA substitutions are highlighted in bold.
  • the stop codon is depicted by an asterisk.
  • the BamHI/Xhol and Hind II l/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Amino acids within boxes encodes the HLA-DR7, HLA-DR53 and HLA-DQ.6 restricted gplOO a-iso CD4 epitope (GTGRAMLGTHTMEVTVYH) in Hl and L3, the HLA-0201 TRP2i 8 o-i88 epitope (SVYDFFVWL) in H2 and the HLA-DR4 restricted gpl00 44.5 9 CD4 epitope in H3 and LI (WNRQLYPEWTEAQRLD).
  • the Hind ⁇ /Afe I and BomHI/Bs/WI restriction sites utilised in transfer of the variable heavy and light regions are highlighted.
  • Amino acids within boxes encode the HLA-DR7, HLA-DR53 and HLA- DQ6 restricted gplOOi 73 -i9o CD4 epitope (GTGRAMLGTHTMEVTVYH) in Hl and L3, the HLA-0201 TRP2180-188 epitope (SVYDFFVWL) in H2 and the HLA-DR4 restricted gpl0044- 5 s CD4 epitope in H3 and LI (WNRQLYPEWTEAQRLD).
  • the Hind ⁇ /Afe I and BomHI/Bs/WI restriction sites utilised in transfer of the variable heavy and light regions are highlighted.
  • Amino acids within boxes represent the HLA-DR7, HLA-DR53 and HLA-DQ6 restricted gplOOi 73 -i9o CD4 epitope (GTGRAMLGTHTMEVTVYH) in CDR Hl, the HLA-0201 TRP2i 8 o-i88 epitope (SVYDFFVWL) in H2 and the HLA-DR4 restricted gpl00 4 4-59 CD4 epitope in LI (WNRQLYPEWTEAQRLD) retained from pVAXDCIB68.
  • Additional epitopes include nested within the gplOO 4 7 i-492 sequence inserted into the H3 site (VPLDCVLYRYGSFSVTLDIVQG) a HLA-A1, B35 and predicted HLA-DP4 epitope.
  • TRP2 177-205 and TRP2 so-91 sequences were grafted into the L2 and L3 sites of the variable light region respectively. These collectively contained an HLA-A2, A3, A31, A33, B35, B44, HLA-DR3 and another potential HLA-DP4 epitope as described elsewhere.
  • the Hind ⁇ /Afe I and BomHI/Bs/WI restriction sites utilised in transfer of the variable heavy and light regions are highlighted.
  • Amino acids within boxes represent the HLA-DR7, HLA-DR53 and HLA-DQ.6 restricted gplOO 173-19 o CD4 epitope (GTGRAMLGTHTMEVTVYH) in CDR Hl, the HLA-0201 TRP2180-188 epitope (SVYDFFVWL) in H2 and the HLA-DR4 restricted gpl0044- 5 s CD4 epitope in LI (WNRQLYPEWTEAQRLD) retained from pVAXDCIB68.
  • Additional epitopes include, nested within the gplOO 471-492 sequence inserted into the H3 site (VPLDCVLYRYGSFSVTLDIVQG), a HLA-A1, B35 and predicted HLA-DP4 epitope.
  • TRP2 177-205 and TRP2 50-91 sequences were grafted into the L2 and L3 sites of the variable light region respectively. These collectively contained an HLA-A2, A3, A31, A33, B35, B44, HLA-DR3 and another potential HLA-DP4 epitope.
  • the Hind ⁇ /Afe I and BomHI/Bs/WI restriction sites utilised in transfer of the variable heavy and light regions are highlighted.
  • Nucleotide and amino acid sequence of the antibody heavy and light variable regions cloned inframe with the human igGl CHl-hinge-CH2-CH3 constant region and human kappa constant region respectively within the expression vector pVAXDC.
  • Amino acids within boxes represent the NYESO- 1158-iss HLA-A24 epitope (LLMWITQCF) and NYESO-1 157-155 HLA-A2-restricted epitope (SLLMWITQC) in CDR Hl and H2.
  • NY-ESO-1 33-111 amino acid sequence PESRLLEFYLAMPFATPMEAELARRSLAQ
  • NY-ESO-1 119-143 PSRLLKEFTVSGNILTIRLTAADHR
  • Hind ⁇ /Afe I and BomHI/Bs/WI restriction sites utilised in transfer of the variable heavy and light regions are highlighted.
  • Amino acids within boxes represent the NYESO-liss-iee HLA-A24 epitope (LLMWITQCF) and NYESO-1 157-155 HLA-A2-restricted epitope (SLLMWITQC) in CDR Hl and H2.
  • NY-ESO-1 83-IU amino acid sequence PESRLLEFYLAMPFATPMEAELARRSLAQ
  • NY-ESO-1 u 9 _ 143 PSRLLKEFTVSGNILTIRLTAADHR
  • the Hind ⁇ /Afe I and BomHI/Bs/WI restriction sites utilised in transfer of the variable heavy and light regions are highlighted.
  • human IgGl constant domain CHl-hinge-CH2-CH3
  • the stop codon is depicted by an asterisk.
  • FIG. 34 C57BI/6 or HLA-DR4 mice immunised on days 1, 8 and 15 with SCIB1 or iSCIBl pDNA via gene gun.
  • Splenocytes analysed at day 21 for I FNy responses to TRP2 180-188 peptide (A-C) or gplOO 44-59 peptide (D) by ELISpot assay. Frequency of TRP2 180-188 response compared in different mouse strains (A).
  • B and C TRP2 180-188 response avidity calculated as peptide concentration which elicits 50% maximal response. Titration curves shown as % maximal response.
  • D frequency of responses to O.lpg/ml gplOO 44-59 peptide in HLA-DR4 mice.
  • FIG. 35 C57BI/6, HHDII, HHDII/DP4 or HLA-DR4 mice immunised on days 1, 8 and 15 with SCIBlplus or iSCIBIpl us pDNA via gene gun.
  • Splenocytes analysed at day 21 for I FNy responses to TRP2 180-188 peptide (A-C) or gplOO 44-59 peptide (D) by ELISpot assay. Frequency of responses compared in different mouse strains (A).
  • B and C TRP2 180-188 response avidity calculated as peptide concentration which elicits 50% maximal response. Titration curves shown as % maximal response.
  • D frequency of responses to lpg/ml gplOO 44-59 peptide in HLA-DR4 mice.
  • Figure 36 C57BI6 mice implanted with B16F1 tumour cells on day 1 followed by immunisation with pDNA SCIB1, iSCIBl, SCIBlplus or iSCI Blplus at days 4, 11 and 18.
  • A tumour growth curves.
  • B comparison of tumour volume at day 18.
  • C overall survival.
  • Figure 37 HHDII or HHDII/DR1 mice immunised on days 1, 8 and 15 with SCIB2 or iSCI B2 pDNA via gene gun.
  • Splenocytes analysed at day 21 for I FNy responses to Nyesol 157-165 peptide (A-C) or Nyesol 119-143 peptide (D and E) by ELISpot assay.
  • Frequency of Nyesol 157-165 responses compared in different mouse strains (A).
  • B and C Nyesol 157-165 response avidity calculated as peptide concentration which elicits 50% maximal response. Titration curves shown as % maximal response.
  • FIG 39 HLA-A2 transgenic or Balb/c mice immunised on days 1, 8 and 15 with SN13 or SN14 pDNA via gene gun.
  • Splenocytes analysed at day 21 for frequency of I FNy responses to RBD peptide pool (A) or avidity by peptide titration to RBD 417-425 peptide by ELISpot assay.
  • Avidity calculated as peptide concentration which elicits 50% maximal response. Titration curves shown as % maximal response.
  • Sera dilutions analysed at day 21 for SI protein specific antibody responses in ELISA assay (C) or for SARS-CoV-2 neutralising antibodies in pseudovirus neutralisation assay (D).
  • Figure 40 RBD (A) and N (B) secretion levels (by sandwich ELISA) in conditioned medium and cell lysates six days after transient HEK293 transfections with respective SN constructs.
  • Figure 41 C57BI/6 (C) or Balb/c (A and B) mice immunised on days 1, 8 and 15 with SNll(small RBD trimer), SN12 (RBD trimer) , SN13 (RBD-Fc), SN15 (RBD monomer) all these constructs also contain the modified Fc-N or whole S pDNA via gene gun. Sera dilutions analysed at day 21 for SI protein specific antibody responses in ELISA assay (A) or for SARS-CoV-2 neutralising antibodies in pseudovirus neutralisation assay (B and C).
  • Figure 42 HLA-A2 transgenic, C57BI/6 or Balb/c mice immunised on days 1, 8 and 15 with SNll(small RBD trimer), SN12 (RBD trimer) , SN13 (RBD-Fc), SN15 (RBD monomer) all these constructs also contain the modified Fc-via gene gun.
  • Figure 43 Real-time binding curves (SPR, BiaT200) of the interaction of RBD-FC and RBD-iFCvl at increasing levels of CD64 captured onto a CM5 chip.
  • Figure 44 A. Healthy donor T cell proliferation responses (20-donor panel). PBMC from bulk cultures were sampled and assessed for proliferation on days 5, 6, 7 and 8 after incubation with: iTvl, Herceptin®, Bydureon® and KLH. Proliferation responses with an SI >1.90 (indicated by red dotted line) that were significant (p ⁇ 0.05) using an unpaired, two sample Student's t-test were considered positive.
  • B Box and whisker plots showing healthy donor T cell responses to the iTvl, Herceptin® and Bydureon®. Chart shows maximum proliferation of CD4+ T cells obtained over the time course. Bars represent the 10-90 percentile. Repeated measures one way ANOVA (Friedman test) using a Dunn's post-test pairs comparison are shown for statistical analysis. **p ⁇ 0.01
  • Figure 45 A. 50 donor panel of healthy donor T cell proliferation responses. PBMC from bulk cultures were sampled and assessed for proliferation on days 5, 6, 7 and 8 after incubation with: iTvl, Herceptin®, Bydureon® and KLH. Proliferation responses with an SI >1.90 (indicated by red dotted line) that were significant (p ⁇ 0.05) using an unpaired, two sample Student's t-test were considered positive.
  • Figure 46 Sera from mice immunised on days 1, 8 and 15 with NP, NPFc or NPFciVl via gene gun analysed at day 21 for N protein specific antibody responses in ELISA assay.
  • the spike chain encodes amino acids 319-541 carrying the N501Y mutation of the Kent variant/lineage B.1.1.7, UK -VOC 202012/01.
  • the nucleoprotein chain encodes amino acids 2-419, which includes the D3L and S235F mutations from the variant, fused inframe with the human igGl hinge-CH2-CH3 iVl where murine igG3 23 AA substitutions are in bold.
  • the stop codon is depicted by an asterisk. Mutations of the Kent variant/lineage B.1.1.7, UK -VOC 202012/01 are in bold and highlighted in grey. The BamHI/Xhol and Hind I ll/Pstl restriction sites utilised in transfer of both chains are highlighted
  • Figure 48 Sequence of pVAXDCSN17; SN17
  • the spike chain encodes amino acids 319-541 carrying the K417N, E484K and N501Y mutations of the south African variant VOC 501Y.V2/B1.351.
  • the nucleoprotein chain encodes amino acids 2-419, which includes the T205I mutation from the variant, fused inframe with the human igGl hinge-CH2-CH3 iVl where murine igG3 23 AA substitutions are in bold.
  • the stop codon is depicted by an asterisk. Mutations of the south African variant VOC 501Y.V2/B1.351 are in bold and highlighted in grey. The BamHI/Xhol and Hind I ll/Pstl restriction sites utilised in transfer of both chains are highlighted.
  • Figure 49 Balb/c mice immunised on days 1, 8 and 15 with SN15 (RBD monomer and N linked to modified Fc (FciVl)) via gene gun. Sera analysed at day 21 for antibody reactivity to Lineage A (Wuhan), B.1.351 and B.1.1.7 SI protein variants in ELISA at reciprocal sera dilutions. EC 5 o values are shown.
  • Figure 50 Balb/c mice immunised on days 1, 8 and 15 with SN15 (RBD monomer and N linked to modified Fc (FciVl)), SN16 (RBD monomer and N linked to modified Fc (FciVl) - B.l.1.7 variant), SN17 (RBD monomer and N linked to modified Fc (FciVl) - B.1.351 variant), or whole S DNA via gene gun.
  • Figure 51 BALB/c mice were immunised with SN15 or SN17 DNA constructs at days 1, 8 and 15 and sera taken at day 21 analysed in pseudotype neutralisation assay against Lineage A or B.1.351 pseudotype virus (A) or live virus neutralisation assay against Lineage A virus (B). Data are readings at different sera titrations and are representative of multiple experiments.
  • FIG 52 BALB/c mice were immunised with SN15 or SN17 DNA constructs at days 1, 8 and 15 and splenocytes taken at day 21 and analysed for T cell responses by I FNy ELISpot assay to RBD or N peptide pools. Symbols represent mean response for individual mice, line represents mean value between mice. Data are collated from multiple independent studies.
  • Figure 53 Balb/c mice immunised on days 1 and 29 with either SN15 (RBD monomer and N linked to modified Fc (FciVl)) or SN17 (RBD monomer and N linked to modified Fc (FciVl) - B.1.351 variant), followed by a booster at day 85 with SN17 DNA.
  • Figure 54 SN15 whole plasmid vector nucleotide sequence (SEQ ID NO: 23).
  • Figure 55 SN17 whole plasmid vector sequence (SEQ. ID NO: 24).
  • Figure 56 SN17 whole doggbyone (dbDNA) vector sequence (SEQ ID NO: 25).
  • Figure 57 iSCIBlplus whole plasmid vector sequence (SEQ ID NO: 20).
  • Figure 58 iSCIBlplus whole doggybone (dbDNA) vector sequence (SEQ ID NO: 21).
  • Figure 59 iSCIB2 whole plasmid vector sequence (SEQ ID NO: 22).
  • Covidl9 peptides were selected based on IEDB database (http://www.iedb.org/) binding predictions for HLA-A*0201, HLA-DR*0101 and HLA-DP*0401 and SYFPEITHI (http://www.syfpeithi.de) binding predictions for HLA-A*0201. Cancer antigen peptides were selected based on published sequences, IEDB database (http://www.iedb.org/) binding predictions and SYFPEITHI (http://www.syfpeithi.de) binding predictions.
  • Peptides (Table 2) were synthesized at >90% purity (Genscript), aliquoted to single use vials and stored lyophilized at -80°C then reconstituted in PBS on day of use.
  • N, SI and His tagged RBD proteins were purchased from Genescript (USA).
  • N peptide pool was purchased from Miltenyi Biotec (UK) and RBD peptide pool was purchased from JPT Peptide Technologies (Germany).
  • the backbone of all of the COVID-19 plasmids pVAXDCSNl-SN14 are derived from the FDA regulatory compliant vector backbone of pVAXl (Invitrogen) for use in humans. All nucleotide sections for insertion were codon optimised for expression in humans.
  • SN1-SN4 contain a murine IgK leader while SN5-15 contain a human IgH leader.
  • nucleoprotein amino acids 2-419 (Accession number YP_009724397) alone or fused in frame with the Hinge-CH2-CH3 domain of the HuIgGl constant domain or the variant Hinge-CH2-CH3iVl were synthesised and flanked with Hind II l/Pstl.
  • the heavy chain was excised using Hind II l/Pstl from the intermediate vectors generated from the first round and replaced with the N sections in the second expression cassette alongside the appropriate S section depicted in Figure 1.
  • the huIgGl constant region of the antibody heavy chain encoding the CH1- Hinge-CH2-CH3 domains (Acc No : P01857 Amino acid 1-330) was replaced with the same section encoding the replaced 23 murine lgG3 residues at the specific sites. This was achieved by synthesis of the nucleotide section encoding CH1-Hinge-CH2-CH3 iVl flanked by Afel and EcoRI. The huigGl constant domain was excised from the vectors and the section inserted in frame with the heavy variable using these restriction sites.
  • pVAXDCSN16-17 (SN16-17) two consecutive rounds of cloning were required.
  • the sections were inserted into the BamHI/Xhol sites of the pVAXDCIB68 (SCIB1) plasmid in direct replacement of the SCIB1 light kappa chain in the first expression cassette to generate two intermediate plasmids.
  • nucleoprotein chain comprising of the human IgH leader, full length Nucleoprotein amino acids 2-419 (Accession number YP_009724397) containing either the D3L and S235F mutations from the Kent variant/lineage B.1.1.7, UK -VOC 202012/01 for SN16 or the T205I mutation from the South African variant/ lineage VOC 501Y.V2/B1.351 for SN17, fused in frame with the improved variant Hinge-CH2- CH3 iVl human IgGl constant domain (where 23 Amino acids have been replaced with murine lgG3 residues) were synthesised and flanked with Hind II l/Pstl.
  • the SCIB1 heavy huIgGl chain was excised using Hind I ll/Pstl from the intermediate plasmids generated from the first round and replaced with the Nucleoprotein section in the second expression cassette alongside the appropriate spike section resulting in SN16 and SN17.
  • mice C57BI/6J, Balb/c (Charles River), HLA-DR4 mice (Model # 4149, Taconic), HHDII/HLA-DP4 mice (EM:02221, European Mouse Mutant Archive), HHDII mice (Pasteur Institute) or HHDII/HLA-DR1 mice (Pasteur Institute) aged between 8 and 16 weeks old, were used. All work was carried out with ethical approval under a Home Office approved project licence. For all the studies mice were randomised into different groups and not blinded to the investigators.
  • Cells including B16 melanoma expressing relevant MHCI and II alleles (described previously [140, 183, 186, 187]), were cultured in RPMI medium 1640 with L-glutamine (2mmol/l) with 10% FCS and appropriate antibiotics to maintain plasmids.
  • HEK293T human embryonic kidney cells ATCC CRL1573 were propagated as described previously [188]
  • Murine splenocytes were cultured in RPMI-1640 with 10% FBS (Sigma), 2 mM glutamine, 20 mM HEPES buffer, 100 units/ml penicillin, lOOmg/ml streptomycin and 10 -5 M 2-mercaptoethanol.
  • Cell lines utilised were mycoplasma free, authenticated by suppliers (STR profiling), and used within ten passages.
  • Transient HEK293 transfection Secretion levels from pDNA constructs were evaluated following transient transfections of Expi293FTM cells using the ExpiFectamineTM 293 Transfection kit (Gibco, LifeTechnologies). Briefly, HEK293 cells in suspension (100ml, 2xl0 s /ml) were transfected with lOOpg DNA and conditioned medium harvested at day six post-transfection. Conditioned supernatant was filtered through 0.22pm bottle top filters (Merck Millipore) and sodium azide added to a final concentration of 0.2% (w/v). Cell pellets were lysed in an appropriate volume of RIPA buffer (Sigma Aldrich, R0278) according to the manufacturer's instruction and clarified by centrifugation prior to analysis.
  • RIPA buffer Sigma Aldrich, R0278
  • mice were immunised with lpg of DNA via gene gun intradermally on days 1, 8 and 15 or days 1, 15 and 29 and responses analysed on day 21 or 35 respectively unless stated otherwise.
  • SARS-CoV-2 spike protein plasmids were generated and cloned, and pseudoparticles generated following the methodology described for hepatitis C virus in [189]. Pseudoparticles generated in the absence of the plasmid were used as a negative control.
  • HEK293T cells per well were plated in white 96-well tissue culture plates (Corning) and incubated overnight at 37°C. The following day, SARS-CoV-2 pseudoparticles were mixed with appropriate amounts of antibody and then incubated for lhr at 37°C before adding to cells.
  • lOOpI Bright-Glo Promega was added to each well and incubated for 2mins or cells were lysed with cell lysis buffer (catalog no. E1500; Promega) and placed on a rocker for 15mins. Luciferase activity was then measured in relative light units (RLUs) using either a SpectraMax M3 microplate reader (Molecular Devices) with the SoftMax Pro6 software (Bright-Glo protocol), or wells were individually injected with 50pl luciferase substrate and read using a FLUOstar Omega plate reader (BMG Labtech) with the MARS software.
  • RLUs relative light units
  • Infection by SARS- CoV-2 pseudoparticles was measured in the presence of anti-SARS-CoV-2 mAbs, tested animal sera, preimmune animal sera, and nonspecific IgG at the same dilution. Each sample was tested in duplicate or triplicate. Neutralising activities were reported as 50% inhibitory dilution (IDso) values and were calculated by nonlinear regression (GraphPad Prism version 7), using lower and upper bounds (0% and 100% inhibition) as constraints to assist curve fitting.
  • CVR-GLA-1 infectious virus (CVR-GLA-1) was obtained from the National Centre For AIDS Reagents, NIBSC, UK.
  • Live virus neutralisation assays were performed using method previously described [190], except that 790 TCIDso/ml of the SARS-CoV-2 virus was added to each serum dilution. Additionally, for some experiments, the sera were diluted down to 1:81,920.
  • V-PLEX COVID-19 ACE2 neutralisation kit from Meso Scale Diagnostics LLC was used to investigate the ability of vaccine-elicited antibodies to block the binding of ACE2 to RBD or whole S proteins.
  • V- plex SARS-CoV-2 Panel 7 multispot plates containing SI RBD and whole S proteins from Lineage A (originally identified in Wuhan) and variant (Bl.1.7, Bl.351, P.l) SARS-CoV-2 strains were blocked, followed by incubation with sera at 1:100 dilution and Sulfo-tagged human ACE2 protein, according to the manufacturer's instructions. Results are expressed as percentage inhibition of ACE2 binding via comparison of sera-incubated samples to diluent-containing wells (absence of inhibition).
  • RBD binding inhibition was assessed using kit purchased from Genescript (USA). In brief, sera from immunised mice at various dilutions was mixed with recombinant HRP tagged RBD protein.
  • ELISpot assays were performed using murine I FNy capture and detection reagents according to the manufacturer's instructions (Mabtech AB, Nacka Strand, Sweden).
  • anti-IFNy antibodies were coated onto wells of 96-well Immobilin-P plate and quadruplicate wells were seeded with 5 x 10 5 splenocytes and final concentrations of lOpg/ml synthetic peptides, lpg/ml recombinant protein or lpg/ml peptide pools were added unless stated otherwise. Plates were incubated at 37°C for 40 hrs in an atmosphere of 5% CO2.
  • NP was detected in conditioned medium and cell lysate (six days post -transfection) using the SARS-CoV-2 NP ELISA kit from Bioss, (cat# BSKV0001) according to supplier's instructions. Quantitation relied on the standard curve with NP standard supplied by the kit.
  • RBD secreted and in cell lysate
  • SARS-CoV-2 Spike neutralising mouse mAb Sino Biological, 40591- MM43
  • HRPO-labelled detection antibody from the SARS-CoV-2 S protein RBD Antibody Pair (Epigentek A73682). Capture antibody was coated at 200ng/well; detection antibody was used at a dilution of 1:1000.
  • PBMCs were isolated from healthy community donor buffy coats (obtained under consent from commercial vendors). Cells were separated by density centrifugation using Lymphocyte separation medium (Corning, Amsterdam, The Netherlands) and CD8+ T cells were depleted using CD8+ RosetteSepTM (StemCell Technologies Inc, London, UK). Donors were characterised by identifying HLA-DR and HLA- DQ. haplotypes to 4digit resolution by HISTO Spot SSO HLA typing (MC Diagnostics, St. Asaph, UK). T cell responses to the neo-antigen KLH (Invitrogen, Paisley, UK) were also determined.
  • PBMC peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • iTvl test sample
  • a reproducibility control well (cells incubated with 0.3 pM KLH), a clinical benchmark control well (cells incubated with 5 pM Bydureon®), a low immunogenicity control (cells incubated with 0.3 pM Herceptin®) and a culture medium only well were also included. Cultures were incubated for a total of 8 days at 37°C with 5% CO2. On days 5, 6, 7 and 8, the cells in each well were gently resuspended by mixing 5x using an electronic pipette and 3 x 100 pl aliquots transferred to each well of a round bottomed 96 well plate.
  • the cultures were pulsed with 0.75 pCi [3H]-Thymidine (Perkin Elmer®, Beaconsfield, UK) in 100 pl AIM-V® culture medium and incubated for a further 18 hours before harvesting onto filter mats (Perkin Elmer®, Beaconsfield, UK) using a TomTec Mach III cell harvester.
  • CPM for each well were determined by MeltilexTM (Perkin Elmer®, Beaconsfield, UK) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer®, Beaconsfield, UK) in paralux, low background counting.
  • An empirical threshold of a SI equal to or greater than 1.9 (SI >1.90)has been previously established whereby samples inducing responses above this threshold were deemed positive.
  • Example 1 RBD protein and N protein secretion from transient HEK293 transfections with pDNA
  • HEK293 cells were transiently transfected with the pDNA using Thermofisher's Expi293 system and protein secretion in the medium evaluated using sandwich ELISAs for the RBD protein and Nucleoprotein ( Figure 16). This analysis indicated that for the RBD protein, the constructs containing the RBD-Fc fusion (SN3 and SN7) , gave the highest secretion; closely followed by the unmodified RBD (SN4, SN5, SN8).
  • Trimeric RBD (SN2, SN6, SN9, SN10, SN11 ) resulted in the lowest secretion levels.
  • Nucleoprotein secretion was highest for constructs containing unmodified NP (SN3, SN4, SN7, SN8). As the latter target is more relevant for T cell responses compared to inducing neutralising antibodies; lower secretion levels (such as seen for the N Fc fusion proteins) resulting in more avid T cell responses are desirable.
  • Example 2 T cell responses to the RBD and N proteins (NP) with pDNA delivered via gene gun to HHDII mice
  • T cell responses to pVAXDC SPIKE RBD + NP SN8, pVAXDC SPIKE RBD v2 TRIMER + NPFC (SN9), pVAXDC SPIKE RBD v3 TRIMER + NPFC (SN10) and pVAXDC SPIKE RBD v2 TRIMER + NPFC iVl (SN11) following three weekly immunisations of HHDII mice with pDNA administered via gene gun were measured.
  • the frequency of the I FNy ELISpot responses to all RBD constructs was measured using predicted or previously identified T cell epitopes and the whole SI protein, the RBD recombinant protein and RBD peptide pool.
  • Example 3 T cell responses to the RBD and N proteins (NP) with pDNA delivered via gene gun to HHDII/DR1 mice
  • T cell responses to pVAXDC SPIKE RBD + NP SN8, pVAXDC SPIKE RBD v2 TRIMER + NPFC (SN9), pVAXDC SPIKE RBD v3 TRIMER + NPFC (SN10) and pVAXDC SPIKE RBD v2 TRIMER + NPFC iVl (SN11) following three weekly immunisations of HHDII/DR1 mice with pDNA administered via gene gun were measured.
  • the frequency of the I FNy ELISpot responses to all RBD constructs was measured using an identified T cell epitope (RBD aa417-425), the whole SI protein, whole RBD protein and the RBD peptide pool.
  • Example 4 Construct containing NPFC iVl generates higher frequency responses to N protein
  • Example 5 T cell responses to the RBD and N proteins with pDNA delivered via gene gun to
  • HHDII/DP4 mice T cell responses to pVAXDC SPIKE RBD TRIMER + NPFC (SN2), pVAXDC SPIKE RBD FC + NP (SN3) and pVAXDC SPIKE RBD + NP (SN4) following three fortnightly immunisations of HHDII/DP4 mice with pDNA administered via gene gun were measured.
  • the frequency of the I FNy ELISpot responses to all RBD constructs was measured using predicted and identified T cell epitopes, the whole SI protein and the RBD protein.
  • Significant responses were seen from construct SN2 to the RBD aa417-425 peptide, RBD protein and RBD peptide pool and from construct SN4 to the SI and RBD proteins ( Figure 20).
  • Example 6 Avidity of T cell responses to the SI protein with pDNA delivered via gene gun is superior from RBD FC and RBD TRIMER constructs
  • T cell responses to pVAXDC SPIKE RBD TRIMER + NPFC (SN2), pVAXDC SPIKE RBD FC + NP (SN3) and pVAXDC SPIKE RBD + NP (SN4) following three fortnightly immunisations of HHDII/DP4 mice with pDNA administered via gene gun were assessed for avidity to SI protein titration.
  • Responses in mice immunised with SN2 and SN3 show significantly higher avidity of responses (p ⁇ 0.0001) compared to those immunised with SN4 ( Figure 21).
  • Example 7 - pDNA immunisation generates high avidity peptide specific responses to the RBD 417- 425 epitope
  • Example 8 Frequency and avidity of T cell responses to the N 138-146 peptide with pDNA delivered via gene gun is superior from NPFC iVl construct
  • mice immunised with SN11 show higher frequency as well as slightly higher avidity of responses compared to those immunised with SN10 ( Figure 23). Showing that the NP FC iVl construct (SN11) has induced higher frequency and avidity T cells when compared to the NPFC construct (SN10).
  • the Ab titres to the SI, RBD and N proteins were compared in sera from mice immunised with the monomer RBD construct, the dimer RBD presented as an Fc fusion protein, the RBD construct as a trimer and the shorter RBD as a trimer both presented as a fibritin construct.
  • Antibodies were assessed in sera at 1/100 to 1/10,000 dilution. Strong reactivity to the N protein was observed in sera from all immunised mice even at 1/10,000 dilution (Figure 24).
  • Antibody responses to pVAXDC SPIKE RBD FC + NP (SN3), pVAXDC SPIKE RBD + NP (SN8), pVAXDC SPIKE RBD v3 TRIMER + NPFC (SN10) and pVAXDC SPIKE RBD v2 TRIMER + NPFC iVl (SN11) following three weekly immunisations of HHDII mice with pDNA administered via gene gun were measured in an ELISA assay. Antibodies were assessed in sera at 1/100 to 1/10,000 dilution. Strong reactivity to the N protein was observed in sera from all immunised mice even at 1/10,000 dilution.
  • Example 10 - pDNA delivered via gene gun elicits virus neutralising antibody responses with similar titre to total antibody measurement
  • Virus neutralising antibodies were assessed in a surrogate neutralisation assay for inhibition of RBD binding to plate bound ACE2 receptor.
  • Sera samples were also tested for virus neutralisation in a pseudovirus neutralisation assay.
  • Sera from SN2, SN3 and SN4 immunised mice were assessed after only two immunisations at day 21 whereas for SN5, SN6, SN9, SN10 and SN11 sera was assessed at termination (day 35).
  • Virus neutralisation was also analysed at different sera dilutions in the pseudovirus neutralisation assay (Figure 26C). Titration data shows that sera from mice immunised with construct SN5 demonstrates a 50% neutralisation titre (ID50) at 1/3517 dilution of sera. Mice immunised with construct SN6 a titre at 1/236 dilution and those immunised with construct SN3 a titre at 1/600 dilution ( Figure 26D).
  • All COVID-19 constructs contained a S protein RBD, either as a monomer a trimer or an Fc fusion protein and an N protein, either as a monomer, an Fc fusion protein or an Fc fusion protein modified to allow non covalent association of antigen-Fc fusion protein at the cell surface (Tables 5 and 6).
  • SN11 which expresses the N protein fused to modified Fc gave significantly better T-cell responses to N protein and to the HLA-A2 epitope N 138-146 than N protein fused to unmodified Fc or to the N protein alone.
  • this construct also gave superior responses to RBD, SI and peptide RBD 417-425 than a similar construct expressing the same RBD construct but N-Fc. This suggests that the modified N-Fc is acting like an adjuvant and activating the APCs to also enhance the T-cell response to other antigens.
  • the best VNAbs were stimulated to the RBD monomer (SN1, SN4, SN5, SN8, SN15), the RBD trimer (SN2, SN6, SN9, SN11, SN12) and the RBD-Fc (SN3, SN7, SN13, SN14).
  • Constructs were produced comparing the RBD trimer to the RBD-Fc and the RBD-enhanced Fc in combination with the Fc modified N protein.
  • the constructs containing either the RBD monomer and N protein fused separately to enhanced Fc regions(SN15) or the RBD and N protein fused separately to enhanced Fc regions (SN14) produced the strongest antibody and T cell responses.
  • the examples above show a vaccine that incorporates the RBD of the spike protein to stimulate neutralising antibodies and T cell responses but also the N protein to induce memory T cell responses that will confer protection against not only COVID19 but also any new emerging coronaviruses as the N protein is highly conserved and rarely mutates.
  • Example 11 pDNA encoding T cell epitopes within CDRs of Fc modified HuIgGl construct (iSCIBl) generate strong T cell responses
  • HLA-DR4 Conventional C57BI/6 or HLA transgenic mice (HLA-DR4) were immunised with pDNA encoding SCIB1 (WO2008/116937 - Figure 28) compared to iSCIBl ( Figure 29) via gene gun and immune responses assessed by I FNy ELISpot assay.
  • Figure 34A shows high frequency TRP2 180-188 responses generated from both SCIB1 and iSCIBl DNA in immunised C57BI/6 and HLA-DR4 mice.
  • Analysis of the avidity of responses by peptide titration reveals iSCIBl DNA immunisation to generate higher avidity TRP2 180-188 specific CD8 responses than SCIB1 DNA immunisation in C57BI/6 and HLA-DR4 mice ( Figure 35B and C).
  • HLA-DR4 mice were also analysed for the frequency of responses to the gplOO 44-59 epitope and show a trend to higher frequency responses in mice immunised with iSCIBl DNA ( Figure 34D).
  • Figure 34D mice immunised with iSCIBl DNA
  • HLA transgenic mice HLA-DR4, C57BI/6 or HHDII/DP4 were immunised with pDNA encoding SCIBlplus ( Figure 30) compared to iSCIBlplus ( Figure 31) via gene gun and immune responses assessed by IFNy ELISpot assay.
  • Figure 35A shows high frequency TRP2 180-188 responses generated from both SCIBlplus and iSCIBlplus DNA in immunised C57BI/6, HHDII, HHDII/DP4 and HLA-DR4 mice.
  • Example 13 pDNA encoding T cell epitopes within CDRs of Fc modified HuIgGl constructs (iSCIBl and iSCIBlplus) mediate efficient tumour therapy
  • mice immunised with SCIBlplus DNA or iSCIBlplus DNA show significantly slower tumour growth than controls (p ⁇ 0.0001) (Figure 36B).
  • Analysis of tumour volume at day 18 demonstrates that immunisation with iSCIBlplus DNA results in slower tumour growth compared to SCIBlplus DNA ( Figure 36B).
  • Example 14 pDNA encoding T cell epitopes within CDRs of Fc modified HuIgGl construct (iSCIB2) generate strong T cell responses
  • HLA transgenic mice HHDII or HHDII/DR1 were immunised with pDNA encoding SCIB2 ( Figure 32) compared to iSCIB2 ( Figure 33) via gene gun and immune responses assessed by IFNy ELISpot assay.
  • Figure 37A shows high frequency Nyeso-1 157-165 responses generated from both SCIB2 and iSCI B2 DNA in immunised HHDII and HHDII/DR1 mice.
  • Example 15 pDNA encoding T cell epitopes within CDRs of Fc modified HuIgGl constructs (iSCIB2) mediate efficient tumour therapy
  • HLA transgenic mice HHDII
  • B16 melanoma cells expressing the appropriate MHCI allele
  • immunisation with pDNA encoding SCIB2 Figure 32
  • iSCIB2 Figure 33
  • Tumour growth and survival was monitored.
  • Example 16 COVID-19 specific T cell and neutralising antibody responses from pDNA delivered via gene gun is superior from RBD Fc iVl construct compared to RBD Fc construct
  • mice were immunised with pDNA containing N protein fused to modified Fc (NPFC iVl) alongside either the RBD domain fused to Fc (RBD FC, SN13, Figure 14) or RBD domain fused to modified Fc (RBD FC iVl, SN14, Figure 15).
  • T cell responses were assessed by I FNy ELISpot assay and showed significantly higher frequency responses to a pool of overlapping peptides from the RBD protein in mice immunised with RBD Fc iVl, SN14 compared to RBD Fc, SN13 ( Figure 39A).
  • Example 17 RBD protein and N protein secretion from transient HEK293 transfections with SN11, 12, 13, 14 and 15 pDNA
  • HEK293 cells were transiently transfected with the pDNA using Thermofisher's Expi293 system and protein secretion in the medium and cell lysates evaluated using sandwich ELISAs for the RBD protein and Nucleoprotein Figure 40.
  • Example 18 COVID-19 specific neutralising antibody responses from SN15 pDNA delivered via gene gun is superior to that from whole S pDNA
  • Balb/c and C57BI/6 mice were immunised with pDNA containing N protein fused to modified Fc (NPFC iVl) alongside either the RBD monomer (SN15), RBD trimer (SN12) or RBD monomer linked to Fc (SN13) or a with a whole S pDNA.
  • Antibody responses in immunised Balb/c mice were assessed by ELISA and showed higher titres of antibodies and total IgG specific for the SI protein in SN15 and SN13 compared to whole S DNA or SN11 immunised mice (Figure 41A).
  • Example 19 COVID-19 specific T cell responses from SN15 pDNA delivered via gene gun are superior to that from whole S pDNA Balb/c, HLA-A2 transgenic and C57BI/6 mice were immunised with pDNA containing N protein fused to modified Fc (NPFC iVl) alongside either the RBD monomer (SN15), RBD trimer (SN12), RBD short trimer (SN11) or RBD monomer linked to Fc (SN13) or a with a whole S pDNA.
  • NPFC iVl modified Fc
  • T cell responses in immunised mice were assessed by I FNy ELISpot assay and showed high frequency responses to a pool of overlapping peptides from the RBD protein in mice immunised with SN15, SN13, SN12 and SN11 pDNA ( Figure 42Ai).
  • Responses in SN15, SN11 and SN12 immunised mice were significantly higher (p ⁇ 0.0001) than those from SN13 immunised mice.
  • the immunogenicity iTvl was assessed in a proliferation assay (3H-thymidine uptake) using CD8 depleted peripheral blood mononuclear cells (PBMCs) from a panel of 20 donors representing the European and North American population, covering approximately 77% of HLA alleles.
  • PBMCs peripheral blood mononuclear cells
  • the T cell responses were assessed on days 5-8 following incubation with iTvl or wild-type Trastuzumab (Herceptin®) and Bydureon® controls ( Figure 44A).
  • the modified Fc construct, iTvl generated a small increase in proliferation in 3/20 donors; this is comparable to the results seen with Abzena's low immunogenicity control Herceptin®, where a small increase in proliferation was observed in 2/20 donors.
  • Example 22 Lack of immunogenicity of the modified iFcvl construct in a 50-donor panel
  • a repeat was performed to extend to a 50 donor panel spanning a broader range of HLA types.
  • the modified Fc construct, iTvl generated a small increase in proliferation in 3/50 donors; this is again comparable to the results seen with Abzena's low immunogenicity control Herceptin®, where a small increase in proliferation was observed in 2/20 donors and a larger increase in 1/50 donors (Figure 45A). Stimulation indices of 2 to 2.4 were seen for iTvl, much lower than those observed with either the positive control KLH or Bydureon® which is known to induce anti-drug antibodies in 45% of patients.
  • Example 24 Antibody responses cross reactive with variant strains are induced by SN15 pDNA vaccination
  • Example 25 Antibody responses cross reactive with variant strains are induced by SN15, SN16 and SN17 pDNA vaccination that also inhibit ACE2 binding.
  • mice were immunised with pDNA containing the RBD monomer (SN15), RBD monomer from B.l.1.7 variant (SN16, Figure 47) or RBD monomer from B.1.351 variant (SN17, Figure 48) alongside the relevant variant N proteins fused to modified Fc (NPFC iVl).
  • Antibody responses in sera were assessed by ELISA to variant SI proteins from Wuhan (Lineage A), B.1.351 and B.l.1.7 virus strains. High titres of antibodies were observed from both SN17 and SN16 specific for the SI protein from lineage A, B.l.1.7 and B.1.351 variants with detectable responses over background at down to 1 in 100,000 sera dilution.
  • Example 26 Sera from SN15 and SN17 immunised mice show virus neutralisation in pseudotype and live virus neutralisation assays.
  • Sera from Balb/c mice immunised with the original Lineage A (SN15) or B.1.351 (SN17) variant vaccines were also assessed in pseudotype and live virus neutralisation tests against the original Lineage A and B.1.351 variants.
  • Sera from mice immunised with the original variant vaccine showed potent neutralisation of the original Lineage A pseudotype, with reduced efficacy against the B.1.351 variant vaccine (ID 5 o values of 6232 and 2137 respectively) ( Figure 51A).
  • Sera from mice immunised with either vaccine showed neutralisation of the B.1.351 pseudotype variant, but little difference was noted (ID 5 o values of 948 and 997, respectively).
  • Example 27 T cell responses are not impacted by variations in the virus strains.
  • mice were immunised with the SN15 vaccine on days 1 and 29 followed by a boost at day 85 with SN17 vaccine.
  • Antibody responses in sera samples taken at days 42, 82 and 98 were examined by ELISA for reactivity to the Lineage A and B.1.351 SI proteins.
  • mice were immunised with only SN17 vaccine. Antibody responses are detectable in both sets of sera down to 1 in 100,000 dilution (Figure 53A).
  • a drop in response titre is seen at day 82 compared to day 42 in both sets of mice although EC 5 o values remain at >1 in 3500 against the Lineage A SI protein.
  • a booster at day 85 with the SN17 vaccine efficiently boosts responses to the Lineage A SI protein by day 98 in both sets of mice suggesting that the SN17 vaccine is efficient at boosting responses primed by both SN15 and SN17 vaccines.
  • Reactivity in both sets of mice is reduced to the B.1.351 SI protein with lower EC 5 o values compared to the Lineage A SI protein and this is more apparent in mice immunised with the SN15 + SN17 boost.
  • the booster with the SN17 vaccine is able to elevate responses in the SN15 vaccine primed group to a similar level seen in mice receiving the SN17 prime.
  • mice immunised with these prime boost regimes were analysed for reactivity to the RBD proteins from the B.1.351 and B.1.617.2 variants by ELISA (Figure 53B).
  • Sera from both sets of mice show higher antibody titres and EC 5 o values to the B.1.351 RBD protein compared to the B.1.351 SI protein.
  • responses to the B.1.351 RBD protein in mice primed with either SN15 or SN17 are efficiently boosted by the SN17 booster shown at day 98 compared to day 82.
  • a higher titre after the booster vaccine is noted in mice primed with the SN17 vaccine.
  • the coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol, 2003. 77(16): p. 8801-11.
  • Virus-specific memory CD8 T cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection. J Virol, 2014. 88(19): p. 11034-44.
  • TRP tyrosinase-related protein
  • IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol, 2010. 11(11): p. 997-1004.
  • HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature, 2009. 462(7269): p. 99-103.
  • Bodanzsky M. and A. Bodanzsky, The practice of peptide synthesis. 1984, New York: Springer Verlag.

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Abstract

La présente invention concerne des acides nucléiques et des peptides codés par ces acides nucléiques. En particulier, les peptides comprennent une région Fc d'IgG1 modifiée et un ou plusieurs épitopes hétérologues, qui peuvent être des épitopes de lymphocytes B ou T. Un acide nucléique selon l'invention peut coder pour un polypeptide comprenant : (i) une région Fc modifiée d'une IgG1 humaine, et (ii) au moins un antigène hétérologue, (a) la région Fc modifiée comprenant au moins la partie de Fc qui est susceptible de se lier au CD64 et/ou au TRIM21, (b) au moins un résidu de la région Fc étant modifié par rapport au résidu correspondant d'un anticorps IgG3 de souris et (c) la région Fc modifiée présentant une avidité accrue pour le récepteur Fc-gamma (FcγR) par rapport à la région Fc de type sauvage correspondante.
EP21769694.7A 2020-08-26 2021-08-25 Acides nucléiques codant pour un polypeptide comprenant une région fc modifiée d'une igg1 humaine et au moins un antigène hétérologue Pending EP4203999A1 (fr)

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GBGB2013385.6A GB202013385D0 (en) 2020-08-26 2020-08-26 Nucleic acids and polypeptides encoded thereby
GBGB2101435.2A GB202101435D0 (en) 2021-02-02 2021-02-02 Nucleic acids and polypeptides encoded thereby
PCT/EP2021/073542 WO2022043400A1 (fr) 2020-08-26 2021-08-25 Acides nucléiques codant pour un polypeptide comprenant une région fc modifiée d'une igg1 humaine et au moins un antigène hétérologue

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US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4263428A (en) 1978-03-24 1981-04-21 The Regents Of The University Of California Bis-anthracycline nucleic acid function inhibitors and improved method for administering the same
AU7757581A (en) 1980-11-19 1982-05-27 United Energy Technologies Inc. Enhanced surface tubing
IE52535B1 (en) 1981-02-16 1987-12-09 Ici Plc Continuous release pharmaceutical compositions
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
JPS5811808A (ja) 1981-07-16 1983-01-22 Niles Parts Co Ltd 方位検出表示回路
EP0088046B1 (fr) 1982-02-17 1987-12-09 Ciba-Geigy Ag Lipides en phase aqueuse
DE3464682D1 (en) 1983-05-09 1987-08-13 Gen Electric Co Plc Cathode ray tube display device
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4615885A (en) 1983-11-01 1986-10-07 Terumo Kabushiki Kaisha Pharmaceutical composition containing urokinase
US7067110B1 (en) 1999-07-21 2006-06-27 Emd Lexigen Research Center Corp. Fc fusion proteins for enhancing the immunogenicity of protein and peptide antigens
GB0102145D0 (en) 2001-01-26 2001-03-14 Scancell Ltd Substances
GB0706070D0 (en) 2007-03-28 2007-05-09 Scancell Ltd Nucleic acids
GB0901593D0 (en) 2009-01-30 2009-03-11 Touchlight Genetics Ltd Production of closed linear DNA
CN111333704B (zh) * 2020-02-24 2021-01-12 军事科学院军事医学研究院微生物流行病研究所 新型冠状病毒covid-19疫苗、制备方法及其应用

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KR20230058081A (ko) 2023-05-02
BR112023002195A2 (pt) 2023-03-14
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